NOMENCLATURE OF ENZYMES

• Formal systematic nomenclature and classification number

EC (for Enzyme Commission)
– class (1 – 6)
• sub-class
– sub-sub-class
» individual number within sub-sub-class

e.g. EC 4.1.1.28 = L-aromatic amino acid carboxy-lyase

 Classification of enzymes by the reaction catalyzed
1) Oxidoreductases:
dehydrogenases addition or removal of H
oxidases two-electron transfer to 02 forming H2O2
two-electron transfer to ½ O2 forming H2O
oxygenases incorporate 02 into product
hydroxylases incorporate ½ O2 into product as -OH and form H20
peroxidases use as H202 as oxygen donor, forming H20

2) Transferases:
kinases transfer phosphate from ATP onto substrate

3) Hydrolases:
(e.g. esterases, proteases, phosphatases, deamidases)

4) Lyases:
(e.g. dehydratases, hydratases, decarboxylases)

5) Isomerases:

6) Synthetases:
frequently linked to utilization of ATP

Introduction to Nutrition and Metabolism, 4th edition, CRC Press,

Boca Raton. Presentation © copyright David A Bender 2007
PROPERTIES OF ENZYMES
`
HOW ENZYMES WORK ?
Factors that affect the activity of enzymes

 pH of incubation or environment
 temperature
 time of incubation
 concentration of enzyme
 concentration of substrate
 covalent modification of enzyme
 inhibitors and activators
 The effect of temperature on enzyme
activity
10 min incubation
Product formed

1 min incubation

0 20 40 60 80 100
temperature ºC
by convention enzyme activity is always measured at 30ºC
Introduction to Nutrition and Metabolism, 4th edition, CRC Press, Boca Raton.
Presentation © copyright David A Bender 2007
 The relevance of Km
Two enzymes “competing” for substrate S
enzyme A P
S
enzyme A
enzyme B X
rate of reaction, µmol /min

120
low Km
100

80

60

40 enzyme B
high Km
20

0
0 100 200 300 400

[substrate], mmol /L
Introduction to Nutrition and Metabolism, 4th edition, CRC Press, Boca Raton.
Presentation © copyright David A Bender 2007
 Inhibition of enzyme activity
 reversible inhibitors
 non-covalent (equilibrium) binding to enzyme
 many are relatively unspecific

 irreversible inhibitors
 bind to enzyme covalently
 many are substrate analogs
 undergo part of reaction
 transition state covalent intermediate does not break down

 mechanism-dependent (suicide) inhibitors

 highly specific for target enzyme
 so-called rational drug design
 based on studies of enzyme mechanism
 Reversible versus irreversible inhibitors as drugs
Reversible inhibitors diffuse onto and off the enzyme
 therefore undergo metabolism and excretion
 dose may be required several times per day
Irreversible inhibitors are covalently bound
 inactivate a molecule of enzyme permanently
 dose required only daily or less often
 (depending on rate at which new enzyme protein is synthesized)
 it may take a long time to adjust the patient’s dose

Omeprazole is used to treat gastric ulcers

 irreversible inhibitor of proton pump in gastric mucosa
 dose required only once per day
 overdose would not matter because the aim is to inhibit acid
secretion more or less completely to permit ulcer to heal

Introduction to Nutrition and Metabolism, 4th edition, CRC Press, Boca Raton. Presentation © copyright
David A Bender 2007
 Inhibition of enzyme activity
The two most commonly encountered types of inhibition are
competitive and noncompetitive
COMPETITIVE INHIBITOR
NONCOMPETITIVE INHIBITOR
REGULATION OF ENZYME ACTIVITY
A. Regulation by allosteric (cooperative)
binding sites
B.
Enzymic measurement of metabolites -
high sensitivity
rate of reaction, µmol /min

120

100

80

60

Excess enzyme
40
and sample diluted so that [substrate] << Km
large increase in product formed
20 for small change in [substrate]

0
0 100 200 300 400

[substrate], mmol /L
Introduction to Nutrition and
Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Enzymic measurement of metabolites - specificity
Cu2O
(red-brown ppt)
Cu++

HC O COOH
HC OH alkaline copper reagent HC OH
HO CH HO CH
HC OH HC OH
HC OH HC OH
CH2OH glucose oxidase
CH2OH
glucose gluconate
O2
H2O2
ABTS (colourless)
alkaline copper will give positive result
peroxidase
with any reducing compound
oxidised ABTS (blue)
glucose oxidase will only react with glucose ABTS = 2,2'-azo-di-
H2O (3-ethylbenzothiazoyl-sulphonate)

Introduction to Nutrition and

Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Measurement of enzymes for diagnosis
Excess substrate, so that enzyme is saturated
limiting factor in product formation
rate of reaction, µm ol /m in

120 is amount of enzyme present

100

80

60

40

20

0
0 100 200 300 400 500 600 700 800

[substrate], mmol /L
Introduction to Nutrition and
Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Measurement of enzymes for
diagnosis
heart damage (myocardial infarction)
 creatine kinase
 aspartate transaminase
 lactate dehydrogenase
impaired liver function
 aspartate transaminase
 alanine transaminase
 alkaline phosphatase
 g-glutamyl transpeptidase
bone disease (including preclinical rickets)
 alkaline phosphatase
prostate disease
 acid phosphatase

Introduction to Nutrition and

Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Plasma enzymes after myocardial
infarction
creatine kinase aspartate aminotransferase lactate dehydrogenase
7 7 7
activity relative to control

6 6 6

5 5 5

4 4 4

3 3 3

2 2 2

1 1 1

0 0 0
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8

days after myocardial infarction

Introduction to Nutrition and

Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Rickets – a disease of vitamin D
deficiency

plasma alkaline phosphatase

 normal children 100 – 300 units /L
 children with sub-clinical rickets > 390 units /L

Introduction to Nutrition and

Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright David
 Key points
Breaking covalent bonds involves an input of energy to excite electrons to an
unstable configuration (the activation energy).
Exothermic reactions proceed with output of heat; endothermic reactions require an
input of energy.
Enzymes catalyze reactions by lowering the activation energy; they increase the rate
at which equilibrium is reached, but do not affect the position of equilibrium. In vivo
reactions are not normally at equilibrium because there is constant flux through a
pathway.
The active site of an enzyme comprises a substrate binding site and a catalytic site;
both are formed by reactive groups in the side-chains of amino acids that may be
some distance apart in the primary sequence of the protein.
Enzymes show considerable specificity for the substrates bound and the reaction
catalyzed.

Introduction to Nutrition and

Metabolism, 4th edition, CRC
Press, Boca Raton.
Presentation © copyright
Enzymes may have non-protein components, coenzymes that may be covalently or
non-covalently bound to the protein and are essential for activity.
Most enzymes show a hyperbolic relation between the concentration of substrate
and the rate of reaction; Vmax is the maximum rate of reaction when the enzyme is
saturated with substrate.
Km is an inverse measure of the affinity of an enzyme for its substrate; it is the
concentration of substrate at which the enzyme achieves half Vmax.
A variety of compounds inhibit enzymes; irreversible inhibitors bind covalently to the
active site, permanently inactivating a molecule of enzyme. Reversible inhibitors may
be competitive with respect to substrate, non-competitive or uncompetitive.
A metabolic pathway is a sequence of enzyme-catalyzed reactions; pathways may be
linear, branched, looped or cyclic.
Enzymes can be used to measure metabolites in blood, urine and tissue samples;
measurement of enzymes in plasma samples is useful diagnostically.