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TWO
CHEMICALS OF LIF'E
::st three
: -er. Also
- i968. A
::1i zati on,
,-- ¡sanism
i:¿* -Hill,
zoa. and
':ical and
H¿ll. Inc.,
:. micro- The organism must synthesize all the chemicals needed to operate, maintain, and
:.ibolism. :eproduce the cell. In the following chapters we investigate the kinetics? energet-
.'-s. and control of the major biochemical pathways for such syntheses. A neces-
.=n Fran-
]ary prerequisite for such studies is familiarity with the reactants, products,
:ersetics,
:::ulation, in reaction networks of the
-'atalysts, and chemical controllers which participate
:¡ng and ,'e11.
The present chapter is concerned with the predominant cell polymeric chemi-
:rcellent, ;als and the smaller monomer units from which the larger polymers are derived.
:inciples The four main classes of polymeric cell compounds are the fats and lipids, the
polysaccharides (cellulose, starch, etc.), the information-encoded polydeoxyribo-
nucleic and polyribonucleic acids (DNA, RNA), and proteins. The physico-
chemical properties of these compounds are important both in understanding
:.TS CO,,
cellular function and in rationally designing processes incorporating living cells.
The various biological polymers may be usefully regarded as being either
repetitive or nonrepetitive in structure. Repetitit:e biolo¡1ical polymers contain one
kind of monomeric subunit: distinctions between different types of the same
polymers are primarily due to differences in molecular weight and the degree of
branching of the polymer chains. The major function of repetitive polymers in
the cell is to provide structures with the desired mechanical strength, chemical
inertness, and permeability. Repetitive polymers also provide a means of nutrient
storage. In the latter function, for example, a 7 M glucose solution can be stored
28 BIoCHEMICAL ENGINEERING FUNDAMENTALS
rli
as the polymer glycogen, a cell polysaccharide reserve, with a concurrent redu--
tion in moiarrty by a factor of 10,000 or greater. As cells may need to store exce:. Í@mil,'
food supplies without seriously disrupting the intracellular osmotic pressur. 1iüüUmt
species. Further, each ol these biological polymers has a fixed molecular weigh: iflFrilllflfl
and monomer composition, and the monomers are linked together in a fixeO. tlmullu rl
boron, aluminum, vanadium, molybdenum, iodine, silicon, fluorine, and tin. rü[fuo
Thus, at least 24 different elements are necessary for life. finflüm,l
|0lnuflm
Carbon 50 Sodium I (
Oxygen 20 Calcium 0.5
Nitrogen t4 Magnesium 0.5
Hydrogen 8 Chlorine 0.5
Phosphorus 3 lron 0.2 utilli '(r
The solvent within which cells live is, of course, water. In addition
to its
.- r.r\el) unusual properties (a high heat of vaporization' a high dielectric con-
bond-
§SL:3. :-:.:. th; ability toionize into acid and base, and propensity for hydrogen
- : . \\ ater is an extremely important reactant which participates in many
- t]113f : --.tne-catalyzedreactions.Also,thepropertieswhichbiopolymersexhibitde- this
r i:ght .,:1j srrongly on the properties of the solvent within which they are placed:
r-re d. .:: provides the means of -urry separation process designs' The biological fit-
i::i t¡lrvater and other common chemicals is discussed by Blum [12]'t
"eti
--:a-.t
.- a --
:heir
:.1 LIPIDS
i- ¡he
1., ,leflnition, lipids are biological compounds which are soluble in nonpolar
-- h. _
- - .\'ents such as benzene, chloÁform, and ether' and are
practically insoluble in
:: can in their chemical structure and
.,. .:er. Consequently, these molecules are diverse
:5 ¿nd
:.eir biological function. Their relative insolubility leads to their
presence pre-
:-.minantly in the nonaqueous biological phases, especially. the
plasma and
fuel stor-
,:-sanelle membranes. Fats, which simply serve as polymeric biological
activity' Lipids
:ri. ero iipids, as are several important mediators of biological and
,1 .-., .onrtjtrrte portions of moré complex molecules such as
lipoproteins
..:osacchafides, which again appear piedominantly in biological membranes of
::ils and the external walls of some viruses'
CH3-(CH2)16-COOH CH3-(CH2)7-HC:CH-(CH'z)7-COOH
Oleic acid
Stearic acid
The hydrocarbon chain is nearly insoluble in water, but the
carboxyl group
a sma1l
: \,ery hyárophilic. when a fatty acid is placed at an air-water interface,
:mountoftheacidformsanorientedmonolayer,withthepolargrouphydrated
'Numbers in brackets indicate the reference listed at the end of the chapter'
30 erocHgvtcAL ENGTNEFTRING FUNDAME¡{TALS
,,i
llllliliii '
Polarcarboxylheadl_-Nonpo1arhydrocarbontail ",lltlll : r:
,,
ilfllllilllll lllllliiiili t.
If11ñlllllll]lr
tlllllllililrüllfi lti-lrlrr ::
ilMl ", -
lllllllllllll ll:L *:
lturtliiit,
,]]llufl:lill , l '
Lipi<J monolayer at an air-water intert)ce Lipid micelle in water flllilllll "* I
Figure 2.1 Some stable configurations of fatty acids in water. rilll I l,Lll '' ..
'iirr
in the water and the hydrocarbon tails on the air side (Fig' 2.1). The san:, i,lil'llll1lll,,,: ". :
phenomenon occurs in the action of soaps, which are fatty acid salts. The soai- tilllll q¡ .r,..
monolayer formation greatly lowers the air water interfacial tension, and tl: llllllllllLLLLll
ability of the solution to wet and cleanse confined regions is greatly increased. ilIllillllllll fl
ilLl,,,"''-
o
tl
Na+ - O-C-(CHr)r-HC:CH-(CH2)?-CH3
A soap: sodium oleatc
These hydrophilic-hydrophobic lipid molecules possess very small solubii--
ties: elevation of the solution concentration above the monomolecular solubilil',
iimit results in the condensation ol excess solutes into larger ordered structur.:'
called micelles (Fig. 2.1). This spontaneous process occurs because the overall fre,
energy of the resultant (micelle plus solution) mixture is lower than that of th.
original solution. The structure ol the micelle is dictated by the favorable increas.
in the number olhydrophobic-hydrophobic and hydrophilic-hydrophilic contact.
and concurrent diminution of hydrophilic-hydrophobic associations. Such inte:-
actions between hydrophilic and hydrophobic portions of the sarre biopolyme:
are also known to favor the folding of such polymer chains into a single preferreü
configuration. This behavior of DNA and proteins will be discussed shortly.
Important as reservoirs of fuel, fats are esters formed by condensation c.
fatty acids with glycerol.
o
ll
CH,OH HO-OC(CH2),,-CH3 qH2o-c-(cH2),,-cH:
I + o
3 H"O il
HO-C-(CH2),,-CH3
I
CHOH +
ll"tlttllltl. -.-
+ o
tl iil'r,l
cHroH HO-OC(CH2),.-CH3 HrO-C-(CH2),.-CH.
Glycerol Fatty acids A fat lliil r I
cHEMICALS op lrrs 31
solubili-
solubility
tructures ,ro,".
E= *o,.,
§{
erall free
.at of the
: increase
E=
contacts
Lch inter-
rpolymer
prelerred
rrtly.
sation of Lipid bilayer
',
."', Cell lnembrane
(¿) (á )
-CH. Figure 2.2 (a) The spontaneous formation of a stable phosphoglyceride bilayer in the aperture be-
tween two compartments fil1ed with water ¿nd lipid. (á) This structure strongly resembles the bilayer
appearance of ce11 membranes in electron micrographs. IElectron micrograph reprinted by permission
,.-CH3 .from J. B. Roberfson, Membrane Models: Theoretical and Real, in "The Neruous System, uol. l: The
Basic Neurosciences," D. B. Tower (ed.), p.43, Rauen Press, New York.l
32 etocHgl\atcAI- ENGINEERING FUNDAMENTALS
Severalphysicalpropertiesoflipidbilayermembranesaresimilartotho:e;rr
membranes have high passive ele:::¡ i&
plasma -.*b*rr"r. notñ tipla and plasma resultant impermeability ol natural rn¿:' rut
cal resistance and capacitance. The ,mfl'
branes to such highly charged species as phosphorylated compounds is largeli i
result of this property. The membrane thereby allows the cell to contain a re=:-
metabolic intermediates, as well as maintaining n tlff"* w
voir of charged nutrients and
considerable difference between the internal and external
concentrations of sr'l lffi!
ffi,r¡m
cations such as H*, Kn, and Na+'
exchange bt-
Other membrane components and their influences on material
tween the cell environmeni and interior will be considered
in Secs' 2'5 and Í -
These barriers and passages for speciflc biochemicals determine
which enter' ¿:s
inside the cell' The'¡
confined, and leave the átalytic ieaction network housed
mass transport regulation functions are critical for life,
and they are obviousll ;''"
major importance in process applications employing cells as catalysts'
"
An intriguing similarity between bilayer lipid membranes and plasma
mer:'-
permeabilities b¡ tl:
branes is tháir ability to be modified in their selective ion
antibioti;'
addition of small amounts of various substances. In particular, several
mulmut
fii ,. .tr those of :.rlations (commensalism, considered further in Chap. 13). Several water-solu-
itrt : .,.:re electri- : ', itamins are known to be necessary for activity of specific enzymes.
lr' - ... -lral mem- Steroids are a class ol lipid biochemicals with the general structure shown in
u: -. .! largely a ; 13o. Of these, a subgroup (hormones) constitutes some of the extremely
- -.,riit a reser- :nt controllers of biological reaction rates: hormones may be effective at lev-
u, *., ntaining a . ..,1 10-8 M in human tissue. Microbes are currently used to carry out re-
tr
r:- :s of smali . ', ely minor transformations of such activc steroids to yield more valuable
'Jucts. For exampie, progesterone can be converted info cortisone in a two-
l.- :, ,-hange be- ::' procesS (microbial, then chemical) (Fig. 2.3b). Further exampies appear in
u'-, I5 and 5.7.
¡, - ::. enter, are
l '. ,-e11. These
- . :liously of
1.. . .:
:: :.,1:n1a mem-
r:. --Iies by the
' : .,, ¡tntibiotics )arrl !tr'roi(i base: ¡crhf ilrort c) r:iopctttrttt¡
iIr:'tiI increase :n,r¡tl¡rcne
r: :',. processes, clll
I - : '.ta¿tl or heat
- :: rLluction of ('-o
I
r H.
..,: .:¡ir nucleic I
rn|r^-+,-\l
.--\L-\
'\-zv
l)rogr stc ro nc
-.. lol normal
.' ti an organ-
-1 :' l. the cell
u1Tr.
:.: microbes in
r .:amin.) The
. :.js by defini-
..-: dissolve in
t,
::-1s. The ulti- "'
. ¡-.ception of llt'-{( ),-('H (('lI
a -r
H.
ll
..-
',
:
:: largely from
-,-^\( \
)
.,,;ln (nicotinic
/^.-)\ I I
: .:iamins A, D,
,,nd or adults. ,...-,--\-''t1
--Js, Yeast, lor ('ltolcsterol
:',sunlight to
:,. microbes in
..:al1_v assisting gure 2.3 Some examples of steroid structure.
34 srocusN4icAl ENGINEERING FUNDAMENTALS
Table 12.15. Evidently, the complexity of the reactant is such that only the ac:-":
of an enzyme, produced by perhaps only one or several kinds of microbes, ca::.:
-:
out the reaction with a useful selecriulry (minimal side-product generation). I
familiar steroid cholesterol (Fig. 2.3c) occurs almost exclusively in membr::',
of animal tissues. Related sterol compounds have been shown to alter cell plas;::.-
membrane permeabilities.
An important food-storage polymer for some bacteria, including Alcalig,':".
eutophus, is poly-B-hydroxybutyric acid (PHB). The repeating unit is
CH, O
rll
-cH-cHr-c-o-
The polymer occurs as granules within the cells. In the absence of sufficieflt ft¡;l
supply, the cell depolymerizes this reserve to yield the soluble, easily metaboiiz.-
B-hydroxybutyric acid. PHB is a possible candidate for large-scale manufactu:.
because it is biodegradable and has properties adaptable to packaging.
]lflllnmuuu,, i:i,,'l*I
The carbohydrates are organic compounds with the general formula (CH2O,.
where n > 3. These compounds are found in all animal, plant, and microbi..
cells; the higher-molecular-weight polymers serve both structural and storag.
functions. The formula (CH2O), is sufficiently accurate to be useful in calculatin-;
overall elemental balances and energy release in cellular reactions.
In the biosphere, carbohydrate matter (including starches and cellulose) er-
ceeds the combined amount of all other organic compounds. When photosvc-
thesis occurs, carbon dioxide is converted to simple sugars C. to Ce in reactions 11 -?:
driven by the incident sunlight (considered further in Chap. 5, bioenergetics'
lllllllllll l,,.J,.;r ' ull
These sugars are then polymerized into forms suitable for structure (cellulose) c:
rll[rr' ,- ) lf|g'
sugar storage (starches). By these processes, radiant solar energy is stored ir
flflrlllrlrriiriirii - j if'liu
chemical form for subsequent utilization. The magnitude of this energy transfor-
{llillflllrlü]Íl :lu:
mation is estimated to be 1018 kcal per year, corresponding to storage of 0.1 .1llllllllllll . lliüt''
percent of the annual incident radiant energy. Much of the annual 1018 kcai
füllflll I L;|. r
stored is of course ultimately released in subsequent oxidation (iargely cellulai - i l,ill
respiration) to carbon dioxide. -,f
illililll r.--LJrlt
I cHEMICALS or llro 35
I
HCOH c:o
I
I I
cHroH cH2oH
o-Glyceraldehyde Dihydroxyacetone
C:O
I
HCOH
nfficient food I I
HCOH HCOH
¡ metabolized I I
CH,OH cH2oH
o-Ribose o-Ribulose
HCOH
tl
HOCH HCOH c:o I
HOCH
I ll
HOCH HOCH HOCH
I
nla (CHrO),,
nd microbial
I
HCOH
ll
HCOH HOCH HCOH
I
and storage I
HCOH
tl
HCOH HCOH HCOH
I
in calculating I
CH2OH
ll
cHroH cH2oH
I
cH2oH
n-Glucose ¡-Mannose o-Galactose D-Fructose
ctllulose) ex-
en photosyn-
e in reactions
ioenergetics). Although D-glucose is by far the most common monosaccharide, other sim-
lcelluiose) or pie sugars are also found in living organisms (Table 2.2). These common sugars
is stored in are all either aldehyde or ketone derivatives. In sugar nomenclature, prefixes
rg1'transfor- indicating these functional groups are often combined with a name fixing the
torage of 0.1 iength of the carbon chain. Thus, glucose is an aldohexose; the notation D refer-
nl 1018 kcal ing to a particular optical isomer occurring almost exclusively in living systems
rgely cellular t see optical activity, Sec. 2.4).
ht in the *
s (T,able 2.2).
36 etocFl¡NarcAL ENGTNEERING FUNDAMENTALS
o-Ribose I)eoxvribose
OH hydrolysis
OH HO
't-/
OH
r-o-GIucose a-»-Glucose
CH,OH CH,OH
,ts".'. ,Fo'.,
,ñt"/eñLz,
t-á" i
n"
-6*
e-1,4-glyclsidic bond
z-Maltose üiftlllÍ '
',s0lllLL ;]];
to maltose, which is formed from two o-glucose molecules, the following dis.--
.Il
charides are relatively common:
lrfllllilllr lul
CH.OH ]llffi - 11 ]t*
o LImÍlil)'
{iilll" .ill
cH2oH
)nents of
als to be
B-»-galactose /i-»-glucose
Lactose
-"t>.-,C>"t)"
r *HzO
)H
{>.,,C>;(>._ a-1,4-glycosidic linkages
Portion of an amylose chain (-OH groups omitted for clarity)
r M. M. Green, G. Blankenhorn, and H. Hart, "Textbook Errors. 123; Which Starch F¡action Is
1ñ-ater-Solub1e, Amylose or Amylopectin?," J. Chem. Educ.,852:729, 1975.
38 etocHrNalcAl ENGINEERING FUNDAMENTALS
glucose:
chain and the 6 carbon on another
CC
i-o.. ,t-or
-. <->.(- <,-¿l-cH, i'' 1'':X':""*'1"
->.{>"<_>o{,}o-
-o\ ,¿-o.. /*o' /l-o'
m
2.2.3 Cellulose ii ,l
o\ B-1,4linkage
The glucose chain of cellulose
\r
-t"-
¡lecular weights
'I;!- :': its use of §-r,4-grycosidic bonds. As shown in Fig. 2.4, intrachain hydro_
rs: :.'nding occurs between the c-3 hydroxyl and the á*yg", in the pyránose
-L:¿ ou-casional interchain hydrogen bonding is
also p.érl"rrt. lfhis hydrogen
it can form gels :{::-::lg makes cellulose chains combine to give crystá[ites, larger
-,:: structures
ertain enzymes, tu are visibie in the electron microscope.
¡- Dextrins are :rr eral different models for the crystalline structure of cellulose
have been
other relatively :":::'-'sed: the essential concepts for our purposes can be gleaned
from the sche_
is derived from :n¿:-¡ riew given in Fig. 2.5. Most of the cérurose
is orgánized into highly or-
:r::i'i .r]'srr7lline regions, in which cellulose chains or
fibris are so tightly packed
rer and muscle -:¿: .\'en water molecules scarcely penetrate. cerlulose is, accordlngiy,
water
rctin in that it L::;'::-b1e. Less ordered portions of the assembry,
called amorphou,
re are typically ;'i- t'pically about 15 percent of the cellulose microstructuie. Theregions, com-
amorphous
*ji,rns are easily hydrolyzed
anches. Glyco- by, for example, acids; the crystalline ón the regions
non. Glycogen :.:-:: hand are much more difficult to decompose.
ling the enteric The cellulose fibrils are clustered in microfibrils which
are often depicted in
::: -r> section as in Fig. 2.5a. Here the solid lines denote the planes
of thá glucose
: ---ding blocks, and the broken lines represent orientations
áf another important
r:: rp of polysaccharides called hemicellulose. Hemicellulose molecules
are found
I,sae to trees, is H6
H
Care two com- ..-HO 3
mount of cellu-
e molecule is a o /s o- - -HO
I 2
seight ranging ^l H zo HH 3 I
H H
\ H
\
n_the 1 and 4 \
brently than in \
H6
ceding one for
ICH,oH H H\ H 6
ricroorganisms, .-to
-\-¡._l-o\--_Ho / \ CH2OH
.oHl, H
,o).frfo-+ lffo-i
I
tits joined by a Fteure 2'4 Schematic view of hydrogen bonding between glucose residues
in cellulose. Also indicated
:i,¡ r\ is d
R r§ a posrron lor
possible position
pu§§ruls for chemlcal
chemical modrrication
modification of celrulose.
cellulose. In methyl cellulose, cellulose
cellurose, ,
(a) (b)
I .., v,'t','*,'
I'; 'r.ti"').t;"
::::J
Figure 2.5 Diagram of tbc
ture of cellulose
( Adapted from K. Múle
Ann. Reu Plant Ph1's-. tati
p. 1. 1967.)
clusters of microfibrih ¡
in wood and other cellulosic materials surrounding
thesestructures are in turn in an extensively cross-linked coating
"""u*¿
lignin.
lignocellulose is often used to
Because of this packaging, the term
see that many types,of 01"11t"^ii*
these materials. tn Tabie--l'2,*'"can
lignocellulose.and that the re
ff['H,ffii; ;";i" significant amounts.of
proportions of cellutose,"il""-i""!r"r1:l:* l'^'1'1.::?::TY""i3:'J;,1'r:';
source
that, in addition to serving as a renewable
ü:##"ñ;'.;;ñ";,these biomass resources also contáin t":':
cellulose feedstock, -1Tl':"r::-""t":'
;tii}ffi,;]r"f¡ #;aterials. For this reason, we shall examine further
properties of hemicellulose and lignin' ------^^:¿:^- 6ñ^ñ6 rtifferenr pr
nl:
amons different
""t.;;;,i;;;;:;;t"h 'u'v iL specific composition
and
are short, branched p"iv*"* ár Penioses Gvlose T!I::.?^1i'1:"^T:
lll,'ti,T"J,'JH:,#]';;; ;;;;;). riá," units, which typicanv contain
24 40 I 8-25
Hardwoods stems 40-55
45 50 25-35 25 35
Softwoods stems 10,30
25,-40 25 50
Grasses
Leaves t5-20 80 85 -0
Cotton seed hairs 80-95 5-20 -0
40-55 25 40 18 30
NewspaPer
60-70 10 20 510
Waste PaPers
from chemical PuiPs
cHEMrcALs or lrpp 41
,r..1: ! : :-::rber of acetyl groups, are linked by 1,3-, 1,6-, and 1,4-glycosidic bonds.
: - ¡hese leatures are ill ustrated in the following representation of hard-
lt .r:i: -:¡nicelluiose:
Jx-x-x- X-X-X-X-X-X-X+
l: 2 3
2 3 3 2 3l
LoA uAAAAA-luo-rro
1
GA
4
M
X: xylose M: methoxy group
A: acetyl group B-1,4 glycosidic bond
-:
GA: glucuronic acid
l-§ D:lsram of the struc- ':::: :!e numbers denote the carbon numbers of various bonds.
' ¡t.-.¡lose microfibrils.
c ',,,: K. Mülethaner.
:: P.-:;,:¡ Phys., uol. 18, H2COH H,COH
_l
I
HC CO
I I
HCOH CH,
t¡f microfibrils, and H2COH
¡"s-linked coating of H,COH| qo
cH:
-cH
:iC
L--n'!9.'
CH 1r
en used to describe - t-é ¿"\
iourass and cellulose ?-\ "-r
HC---l't
--r'\ HCOH"COH
_t 'l
r31-'-:-:1: :r ':
Glucuronic acid has not been
outu't'¿ :r ::r- -- 1:' ': ' -: tffiü
family of uronic acids-ilñ u" :-- -- -' -'-' - --"; {s
Lignin i, u porvpl''ry""i4:;r"i
structure for spruce ú;j;";
Fis'.2'6'*: q*,L"1:1'":T:::'l".l,ii;:,- '' ,-r'
arrangei:.: ffl
rhis random
:,"T"Ji:liJr";;5',il;J;;netty or *,is
material. '*
and enzymatic att¿c¡' : 'r;d$
ferent building blocks Xi.i"?r,
..rirtt "fr"-i"ul
brieflyexamine,o*.oi.iñ",7í,iruul.techniquesinChap.4.Inc[tl.jng.-
viewofbiomasschemistryandstructure,weshouldnotethatligntn-:..:'ui-"lll;
and feedstocks'
pott"tiutiy'i"t"t"'ti'g chemicals
are themselv",
AND DNA
23 FROM NUCLEOTIDES TO RNA
Theinformationalbiopo|yrnerDNA,(deoxyribonucleicacid)containsall::.:.1.]1...;
at ':''' rrlr
divide' each daughter cell receives
hereditary information]íVnt" ""tt'
which allows offspring to resemble Pr::: *i'"
E
complete copy of it' p;t;;;;-;Ña' is found in:::
which DNA póssesses
form and operation' ü;];;;;tion mechar:'': tr¡
along the porymir chain. The
quenceof the subunJ;;";;,td"s cr;': u¡r
of DNÁ rlplication in the ce11
information transfer ';;1;; ii*ing controls' The '-:'r,'
discussed in Chap' o,?á'g
with other aspects ofcellular coded::' :"¡c
DNA polymer' and the information
chemistry, the structurl"oi-tt"
subunit sequence are now considered'
Carrier, and Coenzymes
2.3.1 Builtling Blocks, an Energy
their derir¿'-- ::'
in nucleic acids' nucleotides and
to compon^ents Ir:il
In addition their presence
interest on their own' Three
are of considerable biological (fr"-:1t-t'-:
acid, (2) ribose .or .d":'vtib::"
up all nucleotides: fij"prr""tp^rr"tL"
'í,iog"roo, base, usually derived from either purln' 'r¡
sugars), and (3) a
pyrimidine.
H H flü
,-C -c-N.,\ rI
N- NZC-CH :;
I II )CH lll
\-N -c=ñ
HC.- HC¡'*tcH ' I 1Íillilü
H
Purine Pyrimidine
.- -,-,rronic acid is
OH
-: :::cu'nt) or even
o
: casing which
l2'
In the model OH H
I I
Ribonucleotides
(general structu¡e)
'
, . *nd in the se-
T--.' mechanism of
. : .-e11 cycle are OH
:.. :... The subunit
.:. t codcd in the (.a)
H
-.: .: (five-carbon ll
H H^u uanrnt
;.::;r purine or Ad.,rir. H
o H'r*,2 H
o
I
H¡C. -". -N-.H Hr ) tl
H:.ra'".
Y
llr Y\'N- *.-
H
u
I
n
^f u-
H H H
Thymine Cytosine Uracil
(b\
.:
-.,,o nucleotide igure 2.7 (a) The general structure ol ribonucleotides and deoxyribonucleotides.
:=. :::rt¡lved. Ribo- (b) The five nrtrog_
..,rs bases found in DNA and RNA
-:;': r,rhile deoxy-
:,l;- biopolymer
44 etocHgNttcAl ENGINEERING FUNDAMENTALS
;;idr.'íúñine (T) is'found only in DNA, while uracil (U), a ciosell :--- r",r +¡rLD m
are stron! :-" ;
pyri-i¿lrl base, is unique to RNA' Both series of nucleotides
¡lllllllr .]§L]
:lül LIIJL* I 'T
tá"urrt" of their phosphoric acid groups'
L
"i;;;tgy
tives of the other nJ.oti¿lt also serve
relatéd functions in the cell's chemistrr '
re nltrogenous
The cyclic form of AMP, so called because of an intramolecular ring involv-
the bases, ade-
--; :he phosphate group (Fig. 2.8), serves as a regulator in many cellular reac-
,-pes of nucleic
,:-.- including those that form sugar and fat-storage polymers. An inadequacy
closely related : :i '-iic AMP in tissue is related to one kind of cancer, a condition of relativelv
t strong acids
- -: :nrroiled cell growth.
In addition to providing components for nucleic acids, the adenine-ribose
dl'es the corre-
--.-'phosphate is a major portion of the coenzymes shown in Fig. 2.9. Enzyme
[or nucleosides !u-:-::ics are considered in the following chapter: it suflices here to note that
r',genous bases. the
tr'it tt1€ is the additional organic moiety which is necessary for the activation of
lor the nucleo-
-.:::.in enzymes to the catalytically useful form. As essentially all reactions within
ophosphate. It
erivatives with
'-h1'droxyl; for
ion ol ATP to
,bs free-energy
og (molar H+ JH OH
P-O-P-O-CH.
I
-
of thermal en- )O
ll
Adcnosirre
rergy transfor- diphosphate (ADP)
ail in Chap. 5,
is. ATP is the
m nutrients or
sport of mate-
phates are the OH OH
,sphate deriva-
ll's chemistry.
\denosine
..,sphate (ATP)
ttr
Ilo-P-O-P-O-P-O
OH
ll -cH.
o
Cyclic ANIP
o:P_o oH
I
OH
Figlre 2.8 The phosphates of adenosine. AMp, ADp, and ATp are important in cellular energy
:.:sÉr processes, and cyclic AMP serves a regulatory function.
46 srocHENatcAL ENGINEERING FUNDAMENTALS
Coenzyme A
NH:
Adenine I
1I
"7c--az\
nt\"-t\,
*Z(
-'.zN¡ o
1m
l
o
I
HO-P-:O
W
,,u
l
o
Ho P-o CH:
I
I
o OH OH aHr-(-cHr
I
UO- P:() 1 I
unit
TH,
ll <
L
reactlve stte HN
H
HOaH lRrboll¡un I
CH:
HO( lt
HCzc\c coNH,
tl
"tl
HOCH
Hl [n CH:
I
H HN
B-mercapto
,N - -N--1 --(--\r-( CH:
I
ethyleneamin.
r) =c/ H-
rlll
H\- cHr
I
-t \^z{:- //l-tH
L
ll
o
H
OH OH SH
l
the cell arc cafalyzed by enzymes, variations of the coenzyme level provide a
convenient short-term method for cellular regulation of actiue enzyme and thrl-i
of the rate of certain intracellular reactions.
\H: CH. OH
k_ -a B:s¡
-'
r 7c az
I
Nta
'iu )
tt\*-c---'z
- :lO o
H H
\ tP:o _g_p:o Schenratic lornr
t.,,n\l
I
CH2oiHl B¿s<,
r,l-P OH
+
| B¡'e.
o L,"r
()
^
t
K)
'\_l/' .,1,1,.1
r\r
\_/ tll
t1 o 5'---.-.---+ 3'
l___l__
oH I{O
L___l I _g_p:o
\ \
-rl:o
_o- cH) o
Vo¡l
.
-CH: cH.'ohi
-a | . B¡'e:
Pañtot henate l.ru\
(
unit
\l\_/
-
t{:
':.:
!
tl
OH
Fugu¡e 2.10 (a) Condensation of several nucleotides to form a chain linked by phosphodiester bonds.
-a - .{rother schematic for a nucleotide chain.
p-mercapto-
li: ethylereamitre
- -rüleus may contain seyeral large DNA molecules. The negative charges on
ii D\A are balanced by divalent ions (procaryotes) or basic amino acids (eu-
:¿r\OteS).
It is important to notice in Fig. 2.10a that the sequence of nucelotides has a
re level provide a :;rection or polarity, with one free 5'-end and the other terminus possessinga3'-
I enzvme and thus .Dd not coupled to another nucleotide. As indicated in Fig. 2.l0b, if is conven-
:i.¡nal to write the nucleotide sequence in the 5' to 3' direction. More a.bbreviated
-lrtations are often used. Suppose in the example in Fig. 2.10b, which contains a
:hosphate group esterified to the 5'-hydroxyl, that Base, is cytosine, Base, is
:denine, and Base3 is thymine. Then the same nucleotide sequence information is
condensation of
!)..
.rtlen written pCpApT, or, even more briefly, CAT.
:onnected between As James Watson and Francis Crick deduced in 1953, the DNA molecul.e
ustrates a possible .'Lrnsists of two polynucleotide chains coiled into a double helix (Fig.2.11). The
:Dorrnously larger: :egular helical backbone of the molecule is composed of sugar and phosphate
ri¡ed in one DNA ¡nits. In the interior of the double helix are the purine and pyrimidine bases. It is
In eucaryotes, the rhe sequence of the four different nitrogenous bases along the polynucleotide
48 elocHENarcAL ENGTNEERTNG FUNDAMENTALS
QH 3lA
(TEN NUCLEO¡:: i
PAIRS PER TL!\
Qo
ffi
w
C in phosphate
C and ¡- in
@
o,
Figure 2.11 Several views of the double-helical structure of DNA. These diagrams show the classica.
Watson-Crick structure [7], also called B-DNA, a right-handed helix in which the planes of the base
pairs are perpendicular to the helix axis. The sketch on the right reveals some geometrical parameter:
of this structure. (Left-hand and center diagrams rc¡trinted with permission /rom M. Yudkin and R.
Olford,"A Guidebook to Biochemistry," p.52, Cambridge Unitersity Press, London, 1971.)
on one strand pairs with thymine on the other. Guanine-cytosine is the second
base pair found in DNA. Figure2J2indictates the remarkabie geometric similar- & 0¡,ri
ity of the two base pair configurations and illustrates the presence of two and J'I
three hydrogen bonds in the A-T and C-G base pairs, respectively.
The two strands of DNA have opposite polarity: they run 5' - 3' in opposite ltm,melü
directions. Combining this information with the base pairing rules, we can infer
from the nucleotide sequence of a single deoxyribonucleotide chain, for example
lüii@
flbuu¡q
5'CGAATCGTA 3' !h
that the corresponding fragment of an intact, double-stranded DNA molecule
fu"up
looks like
]M
r@l mü
5'CGAATCGTA 3' lruülüilu ,l[il
3' GCTTAGCAT 5' ü@Ú!m
dü@!
Similarly, if we have been given only the sequence rW
5' TACGATTCG 3' rÜ,rÑwÚa
CHEMICALS OF LIFE 49
T:r mine
F.----. ss¡ ____l
Adenine
É,
g tttilttttiltilt
ililtt u __ /
( ts-. roA__J \
_ n ttttttt
.'\ ,! tttttilttiltil
?
na
.oo7 H
§-,/
-a),/
----i{'o'
Cr rosine
H ,L--.....-:
s:,t.-==1
,\
-- H il t t t t t t t t t
itrLrs the classical .)l t t t t tttt t
tI a.
:ianes of the base \
rc:::cal parameters \--'t^o-----r
lil,r
il tiltt ,,,,,r,,lllH__
.ll. \'udkin anrt '^ t il ttt
tiltt n __
n/
!
R. ,/
,\\
\\ 'ó44-
'-!
o
itrr ribonucleic
ttttt
I tt t t t
t t t I tt
t tt *=- l,
¡r control and t'/
^v ro.8A
H
7¿r¿
netric similar-
|j:;
,-,::¿-ilijt:-?Ti,*'::":]:Í.:'oj:l'*;lfa i;-g,tyy,*,,ureandFunctíon:'2ded.,p. t4t,by
e of two and '"Í 9*,:'!-!.) ''*1,-'iíifir*;:;;," il",;::i:'i,,l"r,l,,ji''il
:i]. ^Í::::,::Í-'l!':^1."Í::':'
-: in opposite
n'e can infer ¡ e would also have deduced
the same doubre-stranded structure. Thus.
- for example ::formational sense, knowledge of one in an
strand,s sequence implies d;
::e ü;il:::i
1o.1Pl-ementary strand. Each strand is a templatefor the other.
This feature of DNA provides the directions
ror synthesi, ái Juugrrter DNA
\.{ molecule -:om a parent DNA moleóule, a proces,
DNA reprication. rfthe two com_
:'iementary DNA strandt u.. ,.puruted "utt"¿
and double helices are constructed from
:ach strand following the. base-pairing
rules, the end products are two new
--ules, each identical to the original Jouble-stranded mole-
DNA and each containing
-trne new strand and one old strand (Fig. 2.r3). Therefore,
uurrpuirirg provides a
-'hemical reader for the, bio.logical coded in the DNA nucleotide se-
-Árug"
"ruence' The overall mechanism depicted in rig.2.13 was
;n experiment in rr"."r.ruy proved in
which E. cori was grown first in u ..¿iu* .ontaining 15N
50 srocHeN4lcAl ENGINEERING FUNDAMENTALS
m
,¡
gM
,,Ú$
nññeilrr
m;Iii¡f, ti
'lum¡C m
rhi
Fisure 2.13 A simPlifred draS'm
m fluc ¡
OÑ,q rePlication As the rL:!E
strands separate, comPlen:rmr¡¡
to Par='- m'
strands are added
sultins,
each
in two daughter n:'- -=¡r¡¡l
r\
identiial to the Parent NÜi::=:'IÚ e fl{k
each daughter molecule conta::-i'
Ñ
( Fron: ''Ñl
.i*n¿
-S,rrr,rr"f.átn the Parent'
{^
JEi üLM
untl Function"' 2d ea-: ñtflEÚ$u
bv Atiel G. LoewY antl ¡'rr'hn
f:----.] Guanine
l;t"r i*t ,¡ur 4* mm
: Cytosine Sieket:¡tz. Copyright O 19óJ'
R¿-:'m¡d lfli¡m0[1, Murl
iott, n¡nrnoit, antl Winston'
Adenine
ly prr*irrio, of Hott, Rineh¿':" nd M!ñiu[ mr
ThYmine e'il4
- Winston.)
-
miM rh
its GC content'
limits. As expected, ,[;',,;* for bN¡. correlates with
cHEMTcALS o¡ urr 5l
:rrmple, E. coli DNA, which is 50 percent GC, has a T* of 69'C, while DNA
':
tm Pseudomonas aeruginosa, 68 percent GC pairs, gives Q : 16"C. GC content
:. different organisms span a wide range from 23 to 75 percent. Base composition
: one p&r&lneter sometimes used in characterizing species.
If a solution of melted DNA is cooled, the separated complementary strands
¡'.11 anneal to reform the double helix. Similarly, if two different single-stranded
iegments of DNA have complementary base pair sequences, these will hybridize
:: lorm a double-stranded segment. Hybridization is a critical experimental tech-
:rque in biochemistry and in recombinant DNA technology (Chap. 6). Before
::r.ing this brief introduction to the physical chemistry of DNA, we should note
::¿t double-stranded DNA can be melted or denatured by addition of alkali or
.--id to ionize the bases. Also, double-stranded DNA in solution behaves hydro-
:', namically like a rigid rod, while single-stranded DNA acts like a randomly
:,:iled polymer.
As mentioned earlier, all of the DNA required for essential functions
¡rowth and multiplication of cell numbers-in a bacterium like E. coli is con-
"¡ined in a single DNA molecule which is a closed circle. This molecule, which
::tists naturally in a highly supercoiled form, is evident in electron micrographs of
:¿cteria as a distinct but somewhat amorphous nuclear region (recall Fig. 1.2).
This large, essential DNA is called the chromosome of the bacterium; this chro-
:iosome is 4.7 million, or 4700kb, base pairs in circumference (kb denotes kilo-
:rses or 1000 nucleotidest).
The DNA in the membrane-enclosed nucleus of eucaryotes is typically di-
'. rded
among several chromosomes, which may be much larger structures than
:he procaryote chromosome. Eucaryotic chromosomes also contain small basic
{ c-rrtrtáIO1OS
proteins called hístones which account for roughly 50 percent of the chromo-
:scenrril-uga- iurrn€s' mass. The nucleoprotein material in the eucaryotic chromosome is called
¿-iL1r tLlols in -itromatin. Eucaryotic cells also contain separate and smalier DNA molecules in
:heir chloroplast and mitochondria organelles.
tu,iing ri hich It is very important to recognize that cells may contain additional DNA
: :he doubie- molecules. Plasmids are relatively small DNAs which are found in many bacteria
c- The possi- and eucaryotes. Bacterial plasmids, for example, are circular DNA molecules
'*'ith sizes ranging from about 4 to 50 kb. Plasmids in nature endow their host
b,e biological
.-e11s with useful but nonessential functions such as antibiotic resistance. In the
s. In further
c'ihcr central biochemistry laboratory and the production process, plasmids assume a central
role: they are important vehicles of recombinant DNA technology. As we shall
rI method in eramine further in Chap. 6, manipulation of plasmids in the test tube and subse-
n bonds and quent introduction of the recombinant plasmids into living cells genetically re-
t\¡ Lr Stfands programs the cells to produce new compounds or grow more efficiently.
trL-niiored b]' Other small DNA molecules may be inserted into living cells by viruses.
hl D\.{ ab- Bacteriophage, or often called simply phage, are small viruses which infect bac-
a'-.e :n overall teria. These can cause problems in large-scale processes utilizing pure bacterial
bich the ab-
,[et:]r' melted r Lengths in kilobases may be converted to linear contour lengths using the factor 0.34 ¡mlkb.
.-Lf,nfent. Fot Also, for a double-stranded DNA, 1 kb equals approximately 660 kdal mass.
52 erocltpNllcAt- ENGINEERING FUNDAMENTALS
T A
A U
C G
G C
The various RNAs which participate in normal cell function serve the purpose of
reading and implementing the genetic instructions of DNA. Messenger RNA
(mRNA) is complementary to a base sequence from a gene in DNA. Each
mRNA molecule carries a message from DNA to another part of the cell's bio-
chemical apparatus. Since the length of these messages varies considerably, so do
the size of mRNAs: a chain of 103 10a nucleotides in length is typical. RNAs are
typically single-stranded. However, intermolecular and intramolecular base pair-
ing ol RNA nucleotide sequences often play a role in the structure or function of
the RNA.
The message from mRNA is read in fhe ribosorme (Figs 1.2 and 1.3). Up to 65
percent of the ribosome is ribosomal RNA (rRNA), which in turn can be separ-
ated in a centrifuge into several RNA species. For example, the E. coli ribosome
contains three different rRNAs denoted 23S, 165, and 55 and with characteristic
chain lengths of 3 x 103, 1.5 x 103, and 102 nucleotides, respectively. Ribosomes
found in the cytoplasm of eucaryotic cells, on the other hand, contain 28S, 18S,
75, and 55 rRNAs. Different still are the ribosomes found in mitochondria and
chloroplasts.
CHEMICALS OF LIFE 53
-
¡ all celis and a From A. L. Lehninger, Biochemistry,2d ed., table 12-3, Worth publishers, Inc.
¡¡nucleotides in ^-:¡ \'o¡k, 1975.
D\{.
The end result of this intricate information transmitting and translating sys-
:::n is a protein molecule. Proteins are the tangible biochemical expression of the
..:rrrmation and instructions carried by DNA. In the terminology of classical
rrtrc€ss control, they are the final control elements which implement the DNA
::ntroller messages to the cellular process. The dynamics and properties of these
: -.ntrollers are considered next and in several later chapters.
r the purpose of
la-s-.e¡g* P¡O :.1 AMINO ACIDS INTO PROTEINS
in DNA. Each
{ t!¡c cellk bio- Proteins are the most abundant organic molecules within the cell: typically 30 to
-0 percent of the cell's dry weight is protein.
i'Cerably, so do All proteins contain the four most
ica-i. RNAs are
prevalent biological elements: carbon, hydrogen, nitrogen, and oxygen. Average
ular base pair- u'eight percentages of these elements in proteins are c (50 percent). H (7 percent).
of o (23 percent), and N (16 percent). In addition, sulfur (up ro 3 percent) contrib-
".r iunction rtes to the three-dimensional stabilization of almost all proteins by the formation
I t"-rr. Up to 65 oi disulfide (s-S) bonds between sulfur atoms at different locations along the
r can be separ- polymer chain. The size of these nonrepetitive biopolymers varies considerably,
'¡ ith molecular weights ranging from 6000 to over I million. The two major types
- ctrii ribosome
h characteristic s.f protein configuration, fibrous and globular, are shown in Fig. 2.14.
ei1'. Ribosomes In view of the great diversity of biological functions which proteins can serve
ntain 28S, 18S, Table 2.6), the prevalence of proteins within the cell is not surprising. A critical
:e¡1e of many proteins is catalysis. Protein catalysts called enzymes determine the
:trchondria and
:¿te of chemical reactions occurring within the cell. Enzymes are found in a
54 uocnrvrcAl ENGTNEERINc FUNDAMENTALS
l¿¡r* l¡ lllhc u
¿
a-Helir
j_i){
Single chain
(myoglobin )
:]
: I T:Jl,r
Antiparallel
H
B-pleated-sheet
Oligomeric protein
containing fou¡ chains
(hemoglobin)
l
Collagen
triple helix
§ t^" :_ :':xl
variety of cell locales: some are suspended in the cytoplasm and thus dispersed ".,1',*X
throughout the cell interior. Some are localized by attachment to membranes or -: "
Regulatory proteins:
1ac repressor Controls RNA synthesis
Catabolite acli\ator protein Catabolite repression of RNA synthesis
Interferons Induce virus resistance
Insulin Regulates glucose metabolism
Bovine growth hormone Stimulates growth and lactation
Transport proteins:
Lactose permease Transports lactose across cell membrane
Myoglobin Transports O, in muscle
Hemoglobin Transports O, in blood
Storage proteins:
Ovalbumin Egg-white protein
Casein Milk protein
Zein Seed protein of corn
fontractile proteins:
Dynein Cilia and flagella
Siructural proteins:
Collagen Cartilage, tendons
Gylcoproteins Cell wa11s and coats
Elastin Ligaments
, ! I globular.
rased on differences in molecular properties, which in turn are partly due to the
us dispersed
:haracteristics of the constituent amino acids, our next topic.
embranes or
criated pro-
Ell. 1.J.1 Amino Acid Building Blocks and Polypeptides
anes. others Ihe monomeric building blocks of polypeptides and proteins are the fl-amino
nall. hairlike
=--ids. whose general structure is
e flagella are
H
rili are impli- I
tissues.
:rent types of
'l --C-'-COOH
H"N
R
hap. 1 1) are
56 srocH¡N,{icAl ENGINEERING FUNDAMENTAT-s
Thus, amino acids are differentiated according to the R group attached to the r T'
carbon (the one closest to the 1r --'
-cooH group). Since there are generally four
different groups attached to this carbon (the exception being glycine, for whic:.
R : H), it is asymmetrical.
Many organic compounds of biological importance, sugars and amino acid.
included, are optically active: they possess at least one asymmetric carbon anj
therefore occur in two isomeric forms, as shown below for the amino acids:
H H
I I
'l 'C---COOH HOOC- --C- NH,
H,N
l-
R R
I form o form
Solutions of a pure isomer will rotate plane polarized light either to the righ:
(dextro- or d-) or left (levo- or /-). This isomerism is extraordinarily importani - -i --
since viable organisms can usually utilize only one form, lacking the isomeriza- -,r¡ (
tion catalysts needed to interconvert the two. The enzyme catalysts themselves 11 ,iil; - l,r : :': ii
are typically capable of catalyzing reactions involving one of the two forms onl¡. It;t"il,IL :- ,,,
This property has been used commercially to resolve a racemic mixture oi qnm' ".-_ _- i
acylamino acids, converting one isomer only, so that the final solution consists I1 .r.,T1.11 :
of two considerably different and hence more easily separable components. Addi- illilAs üi -i lrl
tional details on this process follow in Chap. 4. As direct physical resolution of '*':u ,t_ :
optical isomers is expensive and slow, such microbial and enzymatic (as well as Á ""- :
other chemical) aids for resolution may become more important. ,l-.
It is intriguing to note that only the l-amino acid isomers are found in mosr s,¿- r, 'f
r¡r
proteins. Appearance of o-amino acids in nature is rare: they are found in the ceii {ü+ '"
walls of some microorganisms and also in some antibiotics. l:lll( ':r: - -"l: ii
The acid (-cooH) and base (-NHr) groups of amino acids can ionize in tlltrlrf , '".-,1 -[ i]
aqueous solution. The amino acid is positively charged (cation) at low pH and lllflrl., "'' ". i,il]ll:'
negatively charged (anion) at high pH. At an intermediate pH value, the amino lllllu rlli |ll l: lilil
acid acts as a dipolar ion, or zwitterion, which has no net charge. This pH is the "1r
isoelectric point, which varies according to the R group involved (Table 2.7). An l1*1r, -., "- , l1 ,*ili[:
amino acid at its isoelectric point will not migrate under the influence of an 1[0N i{r:* !i-J- .i:
applied electric field; it also exhibits a minimum in its solubility. These properties lllllüÍlilrr^: . l-.".llll ¡,1
of amino acids allow utilization of such separation techniques as ion .*"hrrrg.. :u',il"*,]mllmj,l
electrodialysis, and electrophoresis for mixture resolution (Chap. 11). mt'.ll,,J: -Jif \
Illustrated in Fig. 2.15 are the 20 amino acids commonly found in proteins. ;¡lilri i: r: -rJitr Tlf
In addition to the carboxyl and amine groups possessed by all amino acids {l]nlJ iiu dÍl ullllL .
(except proline), the R groups of some acids can ionize. several of the acids Íllllllllll'lLu -;,l[ "iI
possess nonpolar R groups which are hydrophobic; other R groups are hydro- üt1miiillJ Irl i..:r:.[. u,]l:[!.J
philic in character. The nature of these side ihains is importÁt in determining r,ffiri[i: - T]l:l,cTl,,
both the ultimate role played by the protein and the structure of the protein l:-J'Ilui!,*
lllü0,(lli:llll
n¿ched to the a ::Lrtein formation, the condensation reaction occurs between the amino group of
e generally four -:e amino acid and the carboxyl group of another, forming a peptide boncl:
r,cine. for which
HOH o
rnd amino acids 3-\
llli-..--..-.-..r1
nric carbon and
C=OH * H-N Ü-o, -H'o,
nino acids: ,/CC
\ ,/-\
R1 R2
H .g...
| .: ll "'-..
H?
I
_C-OH
' - )>q-c---'-
Rí I
* -,'91' *,
1,,'
H"H
ther to the right
Larilr' important Since the peptide bond has some double-bond character, the six atoms in the
¡ the isomeriza- :ashed-box region lie in a plane. Note that every amino acid links with the next
'':a a peptide bond, thus suggesting that a single
[1'sts themselves catalyst (enzyme) can join all the
tu.o forms only. ¡ubunits provided that some other mechanism orders the subunits in advance of
mic mixture of -ptide-bond formation (Chap. 6).
r-.lution consists The amino acid fragment remaining after peptide-bond formation is the res-
nponents. Addi- "ii¿e. which is designated with a -yl ending, e.g., glycine, glycyl; alanine, alanyl.
al resolution of The list of a sequence of residues in an oligopeptide is begun at the terminus
ratic (as well as --..ntaining the free amino group.
Polypeptides are short condensation chains of amino acids (Fig. 2.16). A little
e tound in most :¡tlection suggests that as the length of the chain increases, the physicochemical
..und in the cell :ha¡acteristics of the polymer will be increasingly dominated by the R groups of
-he residues while the amino and carboxyl groups on the ends will shrink in
ds can ionize in .:rportance. By convention, the term polypeptide is reserved for these relatively
at low pH and ':rort chains. These molecules have considerable biological significance. Many
atrue. the amino llrrmones such as insulin, growth hormone, and somatostatin are polypeptides.
. This pH is the Larger chains are called proteins, with the diffuse dividing line between these
tTable 2.7). An :"ro classifications ranging from 50 to 100 amino acid residues. since the average
influence of an nolecular weight of a residue is about 120, proteins have molecular weighis
.ltese properties -:rger than about 10,000, and some protein molecular weights exceed 1 million.
s'ion exchange, Determination of the amino acid content of a particular protein or a protein
II r. ::rirture can be accomplished in an automated analyzer. Total hydrolysis of the
rnd in proteins. ¡rotein can be achieved by heating it in 6N HCl for 10 to 24h at 100 to 120"c.
LiI amino acids \11 the amino acids with the exceptions of tryptophan, glutamic acid, and as-
al of the acids :artic acid are recovered intact in the hydrochloric form. These can then be
¡ups are hydro- .
.eparated and measured. Alkali hydrolysis can be used to estimate the trypto-
in determining ¡han content. The result of such experiments on the proteins from E. coli, an
t-¡f the protein -ntestinal bacterium, are listec in Table 2.8. From these and other data, it has
:een found that not all 20 amino acids in Fig. 2.15 are found in all proteins.
mino acids. In \mino acids do not occur in equimolar amounts in any known protein. For a
58 grocu¡r'rcAt- FTNGINEERING FUNDAMENTALS
3.+ [ ¡ clic
C}] CII.
H- Cllr-
\\(.II- CH-CH.
C-CH.-
ll
c
,/
H:
,/
ullr
-
H
Cflr- S CI I. ('llr
- - -
Methionine Phenylalanine
(Met; M) (Phc; F)
cHr- cH.-c H
I
-
CII]
Isoleucine
(Ilc: I)
Figure 2.15 The 20 amino acids found in proteins. Shown below each amino acid is the correspond
ing three-lettcr abbrcviation and onelcttcr symbol.
4 Cyclic
Ho-cH2- ?n cH:- c
cH3-cH- =
:.
Scrine Threonine
Tyrosine
S)
(Ser; (Thr; T) (Tyr: Y)
NH, NH,
C-CH. C-CH,-CH.-
// // =
o' ()
E Asparagine Glutamine
(Asn; N) (Gln: Q)
a
o
T -o -o c
!
,C-CH.-
o'
,C_CH.-CH.-
()
' H3 N-(CHl )4-
C
('=c_cllr_ ,2
H,N-(
-il -NH-(( H, r,- *HN .5
NH
NIIi
\,
H
:::spond- Arginine Histidine
(Arg: R) (His; H)
H1
o
-(:
HS-cH2- 9-\
I C-C
//
c- /\ HO-\ Others
t{
Cysteine Proline
(Cys; C) (Pro; P)
(entire molecule)
F:sure 2.15 (continued)
: '.in protein, however, the relative amounts of the various amino acids are
- r:3d.
-{.mino acids are not the onry constituents of all proteins. Many proteins
-ritain other organic or even inorganic components. The other part of these
- 1:iugatedproteinsisaprostheticgroup.Ifonlyaminoacidsarepresent,theterm
'',tle protein, already introduced above, is sometimes used. Hemoglobin, fhe
r'" gen-carrying molecule in red blood cells, is a familiar example
of a conjugated
::')tein: it has four heme groups, which are organometallic complexes
--n, A related smaller molecule, containing
myogrobin, has one heme group. Each molecule
:he enzyme L-amino acid oxidase, which catalyzes deamination
of a number of
-.nino acids, contains two FAD (Fig. 2.9) units. As discussed above,
the pros-
-':ic portion ol ribosomes is RNA.
60 eroct{EN4lcAt- ENGINEERING FUNDAMENTALS
ul *
.t ti',: I ; F.- t¿rll
coo- HNr
.t-5 I IN
l_/
coo CH,
t-
I
CH. H O CH,H
t'
'+"H OCH,HO
ll l' lH t'I H ll ll l' I H 3-¿
H3N \ I ..c\n- .-[- C.-*- 9 .-C-N.-¿ ,.Cod
IHHoÍr,H
!.-6.-N.-[-c-"-
lHll I
Í-a-*
Hll I lHll I
CH, H O CH.
I
o
CH,
t'
CH,
t-
CH,
t-
CH. - --l Frlrn"¡inn
t-
CH.
t'
NH
",t'
| - l0 ll
lr L, ^",.
NH.'
H,N -C--i-
-NH,
rrl- : --:: ¡li
Cly-Asp-Lys-G lu-Arg-H is-Ala
:- :- - - v I
Figure 2.16 A hypothetical polypeptide, showing the ionizing groups found among the amino acids. ,r; *:--: ---
Notice that the listing sequence for the amino acid residues starts at the N-terminal end. Numbers , . - .: ]
near each ionizing group denote the corresponding pK value.
O I
CH. 4. Quaternary How different polypeptide chains fit together; structure stabilized
by same
forces as tertiary structure
* * As a\T -, ---- :
¡\. *..
Tl§
}. ^16
-''riirrril:':::a
^¡c
o.o*á
tC/ O ..tr
\^// [&
*ffi
r!(§B;,.. ,,,§tt'
ftr 6* cly
( *§o_ **6n*
á ; ^,J.--**B'n*; l -t.l Threr-.I
ffif" Gry -*70 ¡¿c ind¡rr ¿n
lkü"** 'f,'lp : ;
, .- re H @
- r it
ro3sr\ dl lgllsÍts
(b)
Figure 2.17 (a) The amino acid sequence, or primary structure, of egg white lysozyme, an enzyme. .,* .u;
(á) The corresponding three-dimensional molecular structure (tertiary structure) of crystalline lyso-
zyme. (By permission of C.C.F. Blake and the editor o.f Nature. From"Structure of Hen Egg-White tr1lll$$, I !i --l
Lysozlme," Nütltre, rol. 206, p. 757, 1965.)
cHEMTcALS or lll'g 63
)-en in trig.2.17b for the lysozyme molecule. This elaborate geometrical structure
.s dictated primarily by the protein's amino acid sequence.
When this fact is coupled with knowledge of the cell's coding system for the
tr- "t,
¡ !*, .mino acid sequence (Sec. 6.1), the significance of primai"y protein structure
¿"; 3n1ergcs. lt is the link between the central DNA controller of the cell and
9X," :he elaborate, highly specific protein molecules which mediate and regulate the
iLi,,Ll jir erse biochemical activities essential for growth and survival.
^»
S.lli, 2.-1.4 Three-Dimensional Conformation:
fr; Arg
60
Secondary and Tertiary Structure
rAl¿-
kr Sec'ondary structure of proteins refers to the structural configuration of the amino
Léit,
-,cid residues which are close neighbors in the linear amino acicl sequence. We
L** :eca1lthat the partial double-bond character of the peptide bond holds its neigh-
-. .,§rtt
rors in a plane. Consequently, rotation is possible only about two of every three
i _r ,;lüiijirr
ronds along the protein backbone (Fig.2.18). This restricts the possible shapes
& ¡5¡ lleu ¡ .ihich a short segment ol the chain can assume.
>--, As already sketched in Fig. 2.14, therc are two general forms of secondary
::nicture: helices and sheets. The configuration believed to occur in hair, wooi,
-,nd some other fibrous proteins arises because of hydrogen bonding between
-:tolnS in closeiy neighboring residues. If the protein chain is coiled, hydrogen
ronding can occur between the group of one residue and the
-C:O -NH
_:roup of its neighbor four units down the chain. A configuration called the
,,-ltelix is the oniy one which ailows this hydrogen bonding end also invoives no
,relormation of bonds along the molecular backbone. Collagen, the most abun-
:ant protein in higher animals, is thought to contain three such ¿-helices, which
-:re intertwined in a superhelix. Rigid and relatively stretch-resistant, collagen is a
:rrrlogical cable. Found in skin, tendons, the cornea of the eye, and numerous
-..l.rer parts olthe body, collagen is important in enzyme and cell immobilization
.,nd in forrnulation of biocompatible materials.
Because of the relative weakness of individual hydrogen bonds, the a-helicai
),ructure is easiiy disturbed. Proteins can lose this structure in aqueous solution
r3cause of competition of water molecules for hydrogen bonds. If wool is
Á I
\tH/
Planar peptide
: -i1 enzyme. N-terminal end
group
C-terminal end
.:-..,1ine lyso-
: E,1Lt-LYhite Figure 2.18 'Ihe planar nature of the peptide bond restricts rotation in the peptide charn. ( From A. L
-.i:ninc¡er." Biot:hemistry." 2d ed., ¡t. 128, Iforfh Publishers, Nev, York, 1975.)
64 stocHpN4tcAI- ENGINEERING FUNDAMFNTALS
-i_
steamed and stretched, it assumes a different, pleated-sheet structure. This
arrangement is the naturally stable one in silk fibers. The pleated-sheet configura-
l,i-r, ':'-
tion is also stabilized by hydrogen bonds which exist between neighboring par-
allel chains. While the parallel sheets are flexible, they are very resistant to -,.'
L l- ' :i
stretching. _::,
Achievement of the secondary structure of flbrous proteins is of importance
lll I
in the food industry. While plant products such as soybeans are valuable sources
rr[ | -1"
of essential amino acids, they lack the consistency and texture of meat. Conse-
quently, in the preparation of "textured protein," globular proteins in solution i*
are spun into a more extended, flbrous structllre.
., :
r lYrL,i._l T)lL¡l
llllll»unrL I l, i utllllfr
l{n 1lI
Figure 2.19 The three-dimensional st¡ucture of myoglobin. Dots indicate the e-carbons of the 121 illlqpmrn' **]lll + I
amino acid residues in this oxygen-carrying protein. A single heme group, with its central iron atom iir,'uüuil tl: .
larger and labeled, is shown in the upper central portion ol the molecule. (Reprinted.fram N. A. llli{l ri\t ¡ il
Edwords and K. A. Hassall, "Cellular Biochemistry & Ph1,siology," p. 57, McGraw-Hill Publishing ilil0ilr
Company Ltd, London, 1971.)
cHEMTcALS or, lrrp 65
^:i.
I- cHz CHz cHu
I
I á
H
.i,,
S
1
CHz
óo ,J,,,,,,,.
,::, Ci=,O
I
I
=
I
I C=C
,
i I
l::t:
:,tt:.,t.CHZ::I::CH:: :
NZ\ = 'Ñü;'¡;
\//
N-H U¡
.,:q..Y [**
= =
ili
I C_OH NH
¡
l
ll
A KXX
I
(cH2 )4 CH, .: CH, rt: ,, :
CH: cF, (cH2 )3
ár\,/e,\^;
Isopeptide
Hydrogen I lonic
Apolar Polar
t ¡r alent Secondary
,::. ol the 121
::::r irOn atOm " :ure 2'20 A summary of the bonds and interactions
which stabilize protein molecular structure.
:,.i t)0m N. A. =' ,,: 'Cell Structure antl Function,', p.210 by Ariel philip
G. Loew1, and Siekeuitz, Copyrig¡ht
l:..'Publishing ' : .969 b.y Ht¡l¡, Rinehart, and Winston, Inc. Reprinted
by pirmission
@)
Ur,i:," ii"nrt. ancl Win_
"¡
66 erocHEN4tcAL ENGINEERTNG FUNDAMENTALs
organics are other obvious candidates for disturbance of the natural tertiary
structure of a protein.
Also important in protein conformational architecture are covalent bonds
which often exist between two cysteinyl residues. By eliminating two hydrogen
atoms, a disulfide bond is formed between two groups:
-SH
o
tl
-NH_C-C_ o
I il
S
I
-NH-C-C_ I
H 2H S
++ t-^disullide
tint ug"
H §
I I
S
I
-NH-C-C-il
-NH-C-C- il o
o
such bonds are found as cross-links within a polypeptide chain, and they some- rullu¡' -i - i" :
times hold two otherwise separate chains: insulin is actually two subchains of 21 n r : ,::
and 30 residues which are linked by disulfide bonds (Fig. 2.21). There is also an
internal cross-link in the shorter subchain. These covalent bonds are more re-
sistant to thermal disruption than the weak interactions mentioned above. How- - -'l , -"...,1
r& I
ever, disulfide bonds can be reduced by an excess of a sulfhydryl compound such *:': il
i,l'l-- "
as B-mercaptoethanol.
'|"'lLl x :'-,l :r.
Protein conformation is a major determinant of protein biological activity.
.,lli." ,1" I ür lll
There is considerable evidence that in many cases a definite three-dimensional
lli: 1 -.1-
shape is essential for the protein to work properly. This principle is embodied, for
LL.
example, in the lock-and-key model for enzyme specificity. This simple model
suggests why the proteins which catalyze biochemical reactions are often highly
specific. That is, only definite reactants, or substrates, are converted by a particu-
lar enzyme. The lock-and-key model (Fig. 2.22) holds that the enzyme has a
specific site (the "lock") which is a geometrical complement of the substrate (the
"key") and that only substrates with the proper complementary shape can bind
to the enzytne so that catalysis occurs. The available inlormation on tertiary
enzyme structure has not only confirmed this hypothesis but also has provided
insights into the catalytic action of enzymes, a topic we consider further in the
next chapter. The direct connection between protein geometry and highly speciflc , 'rl It
interactions with other reagents is also well established for permeases, hormones.
antibodies, and other proteins.
When protein is exposed to conditions sufficiently different from its normal
*.J :r :-¡
;llii|ll]lflr|rui1'
biological environment, a structural change, again called denaturation, typically ,:tltfllluutL ' L;1, Juli]: i1
leaves the protein unable to serve its normal function (Fig.2.23). Relatively smali
changes in solution temperature or pH, for example, may cause denaturation.
cHEMTcALS op nrr 67
:ral tertiary
Ser-Leu-Tyr
tclr-NH"
alent bonds Jv, -vut
:r1 h)'drogen IT'' (INTRA)$
I
I
Leu
Gly-Leu-Val- Glu-Glu-CYs I
Glu
i" I
Asp-NH,
iN" T', T'
Val-Aso--GIu-His-Leu
Tyr-Leu -Val-Cys-Gly-Glu
Ala-Lys-Pro
J the.v some- ligure 2.21 Disulfide bonds link two peptide subchains, denoted A (21 residues) and B (30 residues),
rcLrains of 21 :i the beef (shown here) and other species' insulin molecules. Note also the intrapeptide disulfide
:nkage between two residues ofthe shorter subchain. (Reprintedfrom F. J. Reithel,"Concepts in
::e is also an
S:ochemistry, p. 237, McGraw-Hill Book Company, New York, 1967.)
¡:e more re-
¿bove. How-
hich usually occurs without severing covalent bonds. If a heated dilute solution
'.r
rpound such
-.1denatured protein is slowly cooled back to its normal biological temperature,
:he reverse process or, renaturation, wtth restoration of protein function, often
ücal activity.
rccurs. As with other transitions, higher temperatures favor the state of greater
-dimensional
intropy (disorder) hence extendedness; cooling favors the greatest number of
mbodied, for
-:rorable interactions leading to compactness and renaturation.
imple model
.-,ften highly
r- rr
b¡' a particu-
nzvme has a
.'r.bstrate (the W ffi W+#
Substrote
ape can bind
: on tertiary
has provided conPlex I
urther in the :-zyme' octive site ,r""
rughly specific
"nrr."
on surfoce
ls. hormones,
m its normal i'.'itre 2.22 Simplified diagram of the lock-and-key model for enzyme action. Here the shape of the
'irrr¡.- typically ..:..rtically active site (the lock) is complementary to the shape ofthe substrate key. (Reprintedfrom
latir eiy small : ,t. Peltzar, Jr. and R. D. Reid,"Microbiology," 3d ed., p. 158, McGraw-Hill Book Company, New
denaturation, '.:.1972.)
68 stocHpN{rcAl ENGTNEERTNG FUNDAMENTALS
*ll¡lrllülllllllrur
Native form I lil
Random coil
ril x.il
lllL .L x
properties, is the one which minimizes the molecule's free energy. While we can- .1t,, r r 'r r:
not yet utilize this concept to predict protein structure and function knowing ailLflltLt, ti
only its amino ,acid sequence, this perspective is helpful in thinking about how .' I' r
proteins work under process conditions (which often differ significantly from the
protein's native environment) or when their amino acid sequence is altered. l
Equipped with this conceptual outlook, we will not be surprised to flnd, for 'll r't,,,i I
example, that an enzyme attached to a solid surface has less catalytic activity qLL [ ]l
than it does in solution. rllrfl lr L,,i:
After placing so much emphasis on the connection between protein structure 'ri','r, n,,,-
and function, we should remember that most of the forces which stabilize protein ilI,
'l
structure are weak, allowing the molecule substantial opportunities to "flex" il'"l :,
, l" ,,
about a characteristic average geometry. Just as the biological function of DNA ,l¡..r,
depends on the separability of its two strands under isothermal, intracellular ilLlll:l,L
conditions, the ability to flex slightly has been implicated in natural function of ll n , 'l
several proteins. From a process perspective, the relative ease of thermally or ll,t, il ,,ifr
llt¡
chemically inducing denaturation of many proteins reminds us that the biochem-
rr
r rr r,liilti i,
ical engineer may operate enzyme and cellular processes only over a fairly nar- lllll nr u ,, r "i r
row range of, for example, pH, temperature, and ionic strength. This knowledge
is also useful in other subjects such as protein recovery (Chap. 11) and sterilizer tl
design (Chaps. 7 and 9). il I
rillii,iil
tr i ,
:"
sequently possess quaternary structure. The forces stabilizing quaternary struc-
,l(llllllfli' Ll --,t
ture are believed to be the same as those which produce tertiary structure. While ;.
,,Iiliil ,
disulfide bonds are sometimes present, as in insulin, most of the proteins in
-:
,.J
CHEMICALS OF LIFE 69
sterilizer r
F-rom C¿:// Structure atul F_unction,2d ed., p.221,
by Ariel G. Loewy and philip
Lopyright O 1963, 1969 by Holt, Rinehart, and Winston. Reprinted by permissián Siekevitz.
ol. Holt.
Rinehart, and Winston.
Evidence to date suggests that the subunit makeup of some protein mole- 1- . E,m, t
cules is especially suited lor at least two important biological functions: control
of the catalytic activity of enzymes, and flexibility in construction of related but
different molecules from the same collection of subunits. Different proteins
known as isozymes or isoenzymes demonstrate the latter point. Isozymes are
different molecular forms of an enzyme which catalyze the same reaction within
the same species. While the existence of isozymes might seem a wasteful duplica-
tion of effort, the avaiiability of parallel but different catalytic processes provides
an essential ingredient in some biochemical control systems (Chaps. 3 and 5).
Several isozymes are known to be such oligomeric proteins; in one instance _a'
isoenzymes are composed of five subunits, only two of which are distinct. This
might be viewed as an economical device since five different proteins may be
.:,, L,
, :elated but \\'e have already seen in our perusal of lipids that cell membranes play vital roles
;:::: pfOteinS -n regulating component fluxes into and out of the cell intcrior. trther parts of
-: -'Zvl110S ÉIf€ :le outer surfaces of microorganisms and tissue cells are also extremely impor-
-:.:1on within ,-rnt. From a naturai biological viewpoint, microorganisms, such as bacteria that
, .::ui duplica- -.re in environments much more diverse and much less controlled than cells in
::::-S pIOVideS .rer tissue in an animal, must possess greater physical rigidity and resistance to
-:. -l and 5). rursting under large osmotic pressure. Accordingly, bacteria have much different
,-:.a lnstance :uter envelopes than animal cells.
r.s:inct. This Looking at cell enveiopes from a biochemicar process engineering perspec-
. :;':S may be '\e. we f,nd severai points of interest. The properties of outer cell surfaces deter-
::ine the cells'tendency to adhere to each other or to walls ofreactors, pipes, and
,3parators. As we shall develop in the following chapters, such phenomena have
::rportant influences in formulation of immobilized cell catalysts, operation of
-.¡ntinuous microbial reactors, and cell separation from liquid. The chemical and
::¡chanicai characteristics of cell envelopes determine the cells' resistance to dis-
. -: ht clearly :-rption by physical, enzymatic, and chemical methods, essential in recoverv of
.:-.lrrunds are .:: t racellular products.
: .mino acid As indicated in part by their opposite responses to gram staining, the enve-
: - ', ide several :pes of gram-positive and gram-negative cells are very different (Fig. 2.24¡. rn
-, :ore detail :,,ch case, the envelope is comprised of several layers, but their disposition, thick-
...:-5 of cells. :-.sses, and composition are quite distinctive. Not shown in these sketches are
a
"::1ns are en- ,riety of proteins embedded in and on the surfaces of the cell membranes; these
.'.:11 be one focus of our discussion of membrane
transport in Chap. 5.
while the envelope of a gram-positive bacterium has a single membrane,
..rere are two membranes ol this type in gram-negative cells. The outer and
--. toplasmic membranes in gram-negative bacteria enclose a region called
the
1. '.ríplasmíc space u periplasm. The periplasmic
space, which may include from 20
-' 10 percent of the total cell mass, contains a number of enzymes and sugar and
::rino acid binding proreins.
Flagellum (helical,
12-18 nm in diameter)
Pilus (4-'35 nm
in diameter)
-,psule
r:,nable thickness) Capsule
:sually 15-80 nm thick) pG (variable thickness)
OM (about 8 nm thick)
PG (about 2 nm thick)
PS (about 7 nrn thick.¡
CM (about 8 nm thick)
positive Gram-negative
:"gure 2.24 Schematic diagrams
of thc cell envelope structure of gram-positive and gram-negative
-.,::eria (cM cytoplasmic membrane, pG
peptidoglycan, pS periplasmic space, oM outer mem-
':.te). (AdaptedJrom R. Y. stainer, E. A. Adelberg, J. L.
In¡yaham, and M. L. wheelis,,,Introducti¡¡n
:it¿ .l4icrobial World," p. 128, Prentice-Hall, Englewootl Cllfs, N.J., 1929.)
72 eIocH¡N,{rcAL ENGTNEERTNG FUNDAMENTALS
This is not yet the end of the story of envelope layers in bacteria. Beyond the
outer membrane of many species is a capsule or slime layer which consists of []rllillll:!Iffi ítrlcMülüI
polysaccharide. In one strain of pneumococci, bacteria that cause pneumonia, the nüll&' Lsrtlrtu¡ttt
sists of lipids, protein, and polysaccharide containing mannose. Moving outward rh,,. üililllllflllllllllll
CHEMICALS OF LIFE 73
e::0
::. ¡acterial I
from this membrane through the periplasmic space toward the cell exterior, u e
find next a cell wall. In baker's yeast, Saccharomyces cereuisiae, the wall contain¡
6 to 8 percent protein including several enzymes, and about 30 percent each, b;.
weight, of ¡¡lucan, o-glucose residues joined with É-1,6 linkages and frequent B-1.-:
crossJinks, and mannan whose D-mannose building blocks are a-7,6 linked with
a-l,2branches. Protoplasts of S. cereuisiae may consequently be prepared using a
glucan hydrolyzing enzyme such as a 1,3 glucanase. This is a common step rr.
protocols for introduction of recombinant DNA molecules into yeast hosts.
The cell walls of yeasts and many molds contain chitin, a macromolecuie
comprised of N-acetyl glucosamine building blocks linked with B-1,4 glycosrdic
bonds-
CH.
CO ,:*
NH
NH cHroH
CO
CH.
Chitin
Animal cells, because they normally live in a well-controlled isotonic envi-
ronment, do not possess cell walls. In their plasma membranes, in addition to
phospholipids and proteins, we find 2 to 10 percent carbohydrate. These sugar
components, which are found on the external membrane surface in all mamma-
lian cells examined to date, are combined with lipids and proteins in the lorm ol
glycolipids and glycoprotelns, respectively. #ry
2.5.2 Antibodies and Other Glycoproteins
Proteins with covalently coupled monosaccharides or short oligosaccharide
chains, fhe glycoproteins, are found in considerable variety in and around eucar)-
otic cells. Several enzymes such as glucose oxidase produced by the mold ,4s-
pergillus niger arc glycoproteins. Collagen, the biological coaxial cable mentioned
earlier, consists of glycosylated protein. Some interferons, potent antiviral com-
pounds, are glycoproteins. In fact, most eucaryotic proteins which are in contact
with or are secreted into the cell environment are glycosylated. Several glycopro-
teins are currently or likely soon will be important commercial products. We
shall examine their biosynthesis and transport properties in Chaps. 5 and 6. r; ¡q¡mw, 1Jil l
]i .. t Lt. 't
Antibodies, primary agents in immunological defenses of vertebrates, are gly- i::
tr r,-l - ')q
cop¡oteins (Fig. 2.26). Cell fusion techniques discussed in Chap. 6 now enable ,lill !-
production of homogeneous antibody molecules in animals or in bioreactors. I
_r lru
This important technology signals greater future commercial importance of anti- I , I f:ilil
bodies for diagnosis, drug delivery, and bioseparations. Accordingly, it is appro- Lllt :' -: llr¡
[
priate to examine briefly the origin, structure, and function of antibodies. it -ü
CHEMICALS OF LIFE t:,,
coo- coo-
(b)
igosaccharide
'ound eucary-
Carbohydrate
¡he mold ,4s- chain
rle mentioned F. unit
¡"tiviral com-
rre in contact
:ral giycopro-
products. We
i and 6. Figwe 2.26 (a) Schematic diagram of the structure of an immunoglobulin. C and V denote constant
rates, are gly- :nd variable regions, respectively, while subscripts identify heavy (I1) and light (L) chains. Disulfide
'oonds
are shown, as is the attachment of carbohydrate groups (CHO) to the heavy chains. The stem
5 now enable
::gion (Fc) and the antigen-binding sites are found at the carboxyl- and amino-termini, respec-
r bioreactors. :rrely, of the heavy chains. Antigen binding specificity and affinity depend on the variable regions of
tance of anti- :oth the heavy and light chains. (b) Diagram of the molecular structure of immunoglobulin G, or
¡". it is appro- :qG. an antibody. (Reprinted by perntissionfrom E. W. Siherton, M. A. Nat:ia, and D. R. Dat:ies, Proc.
\,ttl. Acad. Sci., t¡ol. 74, p. 5142, 1977.)
¡odies.
76 rtocn¡ir¡rcAL ENGINEERiNG FUNDAMENTALs
In the previous sections we have reviewed the major smaller biochemicals and
the biopolymers constructed from them. Although the relationships between
chemical structure and cellular functions have already been emphasized, it will
provide a useful perspective to reconsider the dynamic nature and spatial inho-
mogeneity of the structures within which such functions occur. As viable cells
grow, the biopolymers of this chapter must be repeatedly synthesized by the cells.
Usually, the nutrient medium of the cell consists of entities such as sugar, carbon
dioxide, a few amino acids, water, and some inorganic ions, but typically signifi-
cant amounts of the biopolymer starting materials are absent. Thus, from these
simplest of precursors, the cell must synthesize the remainder of the needed ; g'rnr ll- '.,-l
amino acids, nucleic acids, lipids, proteins, etc. The energy-consuming processes
inherent in precursor synthesis and biopolymer formation are considered in
Chap. 5.
There are many suprqmolecular assemblies within the cell. We have already
seen that the cell membrane is a grand collection of a range of molecular varie-
ties. The ribosomes are a separable combination of several different proteins and
nucleic acids. In many cases enzymes catalyzing a sequence of chemical reactions
are maintained in close proximity, presumably to maximize utilization of the
reaction intermediates. The organelles such as mitochondria and chloroplasts
represent the final level of organizational sophistication before the cell itself. The
CHEMICALS OF LIFE 77
The cell
l
I
()rga n r.llc s
¡\ucleus
Nlitochond ria
Ch Ioroplasts
I
I
I
Sup rantolecular I
rssemblies Enz¡-rne conrplexes
(parti.lc wcight Ribosomes
106-i0e) Contr¡ctile sr.stcn¡s
.::ino-
1: site Protcins Poly- Lipitts
.r..[rr,i..
:.-.¡ins.
1
': ma\' \JOR TOPICS OF
C I]-{PTL R 2
ttl
tl
ttt
Antino Simple
t
Fattv
.,...1, \ilJJr\ ¡.1.1..
.! and ++ir
\r
'_"\'een a Keto Phospho- Acetate.
¡cids il) n¡vate, ¡I¿rlonate
.: ri'iii nalrte
,nho-
. .el1s
:.e11s.
::bOn
P¡ecLrrsr¡rs front
::nifi- lhc c¡rirr¡nntc'nt
CO:
: hese (nrol st 1t--1.1 ) H:O
':.'ded N2
Figure 2.27 Asoending order of increasrng complexity and organization of
;isses the biomolecules described
:: this chapter. (From A. L. Leltninger,,,Biochemistrlt,,. tst ed., p. 19, Worth publi.chers,
':J in N.y. 1g70.)
Jifferent levels of complexity, from elemental components to the cell, are pre-
.:eady :ented in Fig. 2.27.
,.
arie- The locations of the various biochemicals of this chapter within a procary-
:s and .¡tic microorganism are summarized in Fig.2.28, and the overall chemical com,
.:ions position of E. coli is outlined in Table 2.12. small molecules like amino
acids and
--- the simpie sugars are dispersed throughout the cytoplasm, as are some larger
:1asts
mole-
;u1es inciuding certain enzymes and tRNA. other biopolymers are
, The ' localized
tuithin the cell or near a surface such as the cell membrane.
78 STOCH¡N,IIcAL ENGINEERING FUNDAMENTALS
Free enzyntes
(protein )
Small molecules
Cell wall (amino acids, nucleotides.
Celi membrane (polysaccharide simplesugars,glycerol,
(lipicl and protein) and peptides) fatty acids, acetate,
pyruvato, keto acids. etc.)
Figure 2.28 A schematic diagram of the intestinal bacterium E. col/, showing the location of the
biomolecules described in this chapter.
PROBLE\IS
I . [htn¡¿'¡. ;rlru:
": I .. :::-:":i
Table 2.12 Composition of a rapidly growing E. coti cellt
HrO 10 18 4x1010 1
RNA 6
165 rRNA 500,000 3x104 1
::Ilahed The ions, nutrients, intermediates, biopolymers and other constituents of the
r:l: cell are linked together through reaction networks (Chaps. 5 and 6). Individual
reactions within such networks are catalyzed by enzymes under the mild condi-
tions conducive to maintenance of the native protein forms discussed earlier.
Membranes, catalyst complexes, and organelles (when present) are assembled
:: ínZymes by the combination of energy-consuming reactions (Chap. 5) and the natural
:::iin) (spontaneous) tendency of their constituent molecules to form spatially in-
homogeneous structures rather than a uniform solute sea.
Chapters 3 and 4 examine the kinetics of enzyme-catalyzed reactions and
enzyme applications. Next, Chaps. 5 through 7 consider energy flows and mass
balances in cellular processes, genetics and the control of cellular reaction net-
works, and flnally mathematical representations of cell-population kinetics. This
collection is the prelude to biochemical-reactor analysis, a major theme of the
balance of the text.
: ol the
PROBLEMS
2.1 Chemical structure (a) Write down the specific or general chemical structure, as appropriate, for
the following substances. Comment on the (possible) functional importance of various groups on the
molecule.
'l
Tryptophan 2t8 33.5
sr,"utL
Lrffilllu"
'nli" iillLll
lilrr
NAD+ 259 18
r llllr
260 ilL lilL I
Riboflavin 31
si tll
NADH 339 6.22
,,n I
Lii
iil ]
(a) An ATP solution in a 1-cm cuvette transmits 70 percent of the incident lighl at 259 nm.
Calculate the concentration of ATP. What is the optical density of a 5 x 10 M ATP solution'l 5
(b) Suppose two compounds A and B have the molar extinction coefficients given in Table
2P2.2. If a solution containing both A and B has an OD (1-cm cuvette) of 0.35 at 340 nm and an OD lllllL llil il i
li
of 0.220 at 410 nm. calculate the A and B concentrations.
r il l Fflil
ilrtrlll 1Ul
Ta.l¡le 2P2.2 Ilt{llLr il ll
t .tfL tlllillll l
ilr il I llll
.ür llnlrulttlqmml
2.3 Molecular weights Sedimentation and diffusion measurements arc oftcn used to provide molec- ü fil llLllfl lil
whereR:gasconstant
T : absolute temperatllre
s : sedimentation coelicient, s -1" xllffur ilil
Verify the molecular weights of the substances in Table 2P3.1 (T: 20'C). Assume p - 1.0.
111 ll
Table 2P3.1
11
ltLn r'ljtLr r
,i
- Teble pI :i(pKcoor*pKNx,)
.: OD . lnuno acids are neutral between p(.oorr and pK*r,.
ió) ln amino acid chromatog*p¡y,"rr"rrr
media are sulfonic acid derivatives
"f."#ji"g."prric
: wirh trre negarve Jri,i" u.ia g.oup,,
ti;:!iü:J§,:T:il:il',",:*:ii*;,"*ru:¡act
- =:l ;: * I *:;l i:l'"',l,
_t:ll
r;
ffi ;; ru'#:.'§ fi:ff
suJfonic acid groups on inert
: :' :Hx IJ ;: :T :,"Jliil*: i,T
support
_ )rlr polystyrene
-;ual quantities of
accessible sulfonic acid and polystyrene
iús :m*Ú-
2.8 Stoichiometrv: aerobic vs. anaerobic species In order to calculate convenienlly nutrienl consump'
tion, aeration, and heat-transfer rates (Chaps. 5, E), a general stoichioriictric equation can be formu-
lated by assigning to the ce11 an apparent molecular formula.
(a) Calculate the cell formula for the growth of yeast on sugar when it is observed to give the
following balance (assume 1 mol of cel1 is produced):
(b) If the above reaction produces 0.30017 "mol" of cell, what cell formula results?
(c) Antrerobic fermentations typically produce a variety of partially oxygenated compounds tn
addition to cell mass. Keeping the cell "molecular formula" of part (b) above, calculate the unknorvn
coemcients for the following typical equation (coefiicients are mole quantities, not mass):
Kinetically, the rates of prooesses (a) and (c) can conveniently be controlled by changing the aeration lll(L]r l. ,,. I I ",ü,:
'u
rate, giving yeast and ethanol at any intermediate desired level (Vienna prot:ess) (J. B. Harrison, r{di.
l,
. Ll {llli,
Intl. Microbiol., 10, 129, l9'71). iLllll llL
i i_ilil
lllllil l'fill:' :,ilL
Table 2P9.1i ,[ I trri ' i
lt ] iLLl
tilllLrx . líilililfl m
Capacitancetechnique 30 150 40 130 lll
n,,l'{l t
O.cm2
Resistance, i02 10s 103 10e .Lllt:]ilü
túxtiitlglr t, I 1ll
lffflüülüürl
(a) Why do these membranes have a common range of thicknesses? (Be as specific as possible,) -,,,.111
I flflL. lnm]]];iiLll
(ó) What cell functions are (possibly) served by membrane resistance and capacitance?
I llLl lI L llllL[:
(c) Compare the breakdown field of the order of 1 V/A with the ionization potential per length t'r r'1lilLlliii,,]l:li {lill
illÍnn
of any Cro or higher hydrocarbon. Comment.
CHEMICALS OF LTFE 83
r:I COltSUmp- 2.10 Concerted-transition model for hemoglobin O, binding The conccrtcd-transition model envisions
::r be formu- .'.n inactive form of protein To in equilibrium with an active form R0:
L
:: '!o give the Rn$To equilibrium constant ¿
In turn, thc active form, which in the casc of hemoglobin has four subunits. may bind one molecule of
:',rbstrate S per subunit:
] LO:
+S +S +S
Ro Rl-R'-R.
S.
Let K,r. denote the dissociation constant for S binding in each step above
- ,:.npounds in
: :ra unknown .. -- LS ll unbound srtesl
Ao'
fbound sites]
.,rd cvaluate the ratio
concentration of S-occupied subunits
J - (otrrl subunit concenlration
2.16 Enzyme structure The three catalytic groups in the 307 amino acid enzyme carboxypeptidase
{1"r,"!¡[\-[';
are arginine 145, tyrosine 248,and glutamic acid2':,O (numbers denote
position relative to the amino-
i
termiÁl resiclue). Calculate the relátive positions of these three residues assuming that the molecule
the real molecule to bring
has only straight, e-he1ix structure. Whafstructural alte¡natives are used in
these three ,.ridu., together, within a few tenths of a nanometer, to effect catalysis?
Assumethat(i)foldingofahydrophobicresiduefromneutralpHaqueousmediumtononpolar . it -r ill
protein interior gives aG - -4 kcal/mol residue and (ii) aG: -5 kcal/mol
polar groups for hydro-
lr 'i 'llu.,Ll::
bond formation between any two unbonded polar groups. Remember that unfolded residues may
ien
hydrogen bond wilh water. r lli I ,l:11,"
sometimes be used to deduce the amino acid sequence of the parent protein.
The following table lists ilt
lill- N-C-
-C-C,
lll:l
li i,
il "rr.t lil
Rloi H
li1
lL : i.. L ii
Table 2P18.1
nLr:lll ,l l'
Agent R, required R, required ".
lL rri
ii , iqil
Trypsin Lys, Arg Any
Chymotrypsin Phe, Trp, Tyr Any
ul L r i,,, ll lLLl:ll_
Pepsin Any Phe, Trp, Try, Leu, Asp, Glu
1r 4 rtil,-l
Cyanogen bromide Met Any
tl
An important adjunct in sequencing studies is the Sanger reaction, in which 1-fluoro-2,4-dinitroben-
DNP-
,ene (poNB) reacts with the NHr.terminal amino acid residue to give the corresponding ¡t,it ir n'
Analysis to dete¡mine
terminal -COOH
group Aspartate
CHEMICALS OF LIFE 85
riooxvpeptidase REFERENCES
l;L1 the amino-
ti ihe molecule 1. L. Stryer, Biochemisty,2d ed., W. H F¡eeman and Company,
rekule to bring San Francisco, 19g1. An advanced
yet highly readable text presenting biochemical principles
in the context of recent molecular
biology.
s phase which 2. M. Yudkin and R. Offord, A Guidebook to Biochemistry,
Cambridge University press, London,
ch. oi the three 1971. A good introductory text which describes the essentials
of biochemistry with a minimum
uations folding amount of detail.
3' A G Loewy and P. Siekevitz, Cell Structure and Function,Holt, Rinehart
and Winston, Inc., New
York' 1969 An excellent introductory short course in biochemistry is
found in part Three. Em-
r.nds. phasis on the experimental verilication of biological theories
and models is an especially strong
feature.
c ro nonpolar 1. A. L. Lehninger, Biochemistry,2d ed., worth publishe¡s, New york, j.975.
An excellent, more
n'Lrps for hydro- advanced biochemistry text. Continued refe¡ence to examples
from cell and human physiology
ri residues may helps the ¡eader
in appreciating, remembering, and organizing the material.
5- A L. Lehninger, Principles of Biochemistry, Worth Publishers, New york, 19g2. A recent text
iequences can written for undergraduates, in the same style as Lehninger's previous
biochemistry book and with
eins table iists a more modern, molecular biology perspective.
6. H. R. Mahler and E. H. cordes, Biorogicar chemistry, Harper
& Row, publishers, Incorporated,
New York, 1971. Greater chemical and mathematical detail than
Lehninger; the sections on ex-
perimental techniques in biochemistry and microbiology go
further in -oá"iíng urd analysis in a
transport-phenomena vein than most other texts.
l. J. D. watson, Molecurar Biology of the Gene,3d ed., w. A. Benjamin,
Inc., Menlo park, califor_
nia, 1976. Although molecular genetics is the main theme, this well-written
classic begins with a
concise presentation of biochemical principles. The significance
of weak interactions and molecu-
lar dynamics in biochemical structure and function is presented lucidly.
5. W. B. Wood, J. H. Wilson, R. M. Benbow, and L. E. Hood,,
Biochemistry,; A problem.s Approach,
2d ed., Benjamin/cummings publishing company, Menlo park,
california, 19g1. concise presen_
tation of biochemistry principres illustrated and amplified by problems
_
9 R M. Dowben, Generar physíorogy. A Morecurar Approach, Harper & Row,answers;.
(and
publishers, Incor-
porated, New York, 1969. A book on cell physiorogy
drawing upon fundamental principles of
physical chemistry: challenging but enjoyable reading.
r' R. w' Dickerson and I. Geis, The structure
and Action of proteins, Harper & Row, publishers,
Incorporated, New York' 1969. A joint project of a biologist
and a sciencá artist, this monograph
I {-dinitroben- provides a refreshing survey of the various protein functions
prrnding DNP- with special .-píor6 on relations
with protein structu¡e. Mosl useful with one of the above references.
. r. J. D. watson, The Double Helix, Athenetm, New yo¡k,
1 96g. The story of the discovery
i/!-e its primary of DNA
structure, a turning point in biology. Also a candid glimpse at
4 Crs.2 Asp,4 the process ofdiscovery in modern
research.
H. F. Blum, Tine's Aruow and Euorution,3d ed., princeton university press, princeton,
N.J., 196g.
'{n inquiry into the biological fitness of elements and chemicals f¡om an evolutionary viewpoint.
Fukui and lshida. Mícrobiar production of Amino,4cfuls, Kodansha Ltd.,
Tokyo, and John wiley
and Sons, Inc', New Yotk, 1972. An interesting survey of devices
to release-mltabolic interme-
diates, in this case amino acids.
É-\'al-GIu-Glu,
t{is-Cys, Glu-
l-.C1s. Ser-Leu