1

Microbial Physiology and Biochemistry

Part 1: Enzymes, intro to Energetics, and Membrane transport
This portion of the course provides the details behind how microorganisms obtain
energy and grow. The act of growth requires the conversion of chemical or light energy
to biochemical energy that the organism can use. There are relatively few ways that
chemical energy can be captured by living organisms and can be used to do work, such as
building new cell material. What you will be learning are mostly all variations on the
same theme. The theme is the coupling of electron transfer reactions from the breakdown
of organic or inorganic compounds (catabolism) to the production of energy or cellular
material (anabolism). Collectively the study can be called microbial metabolism. In
order to couple chemical reactions to energy production a living system must have a way
of controlling these reactions. You will want to review your thermodynamics because I
use thermodynamic principles in the explanations.
In most cases, although the free energy change of the reactions are negative,
indicating that energy will be released during the reactions, these reactions are not
spontaneous, that is they have a high activation energy. This must be overcome by the
living system in order to release the energy. This is accomplished by the use of enzymes.
Enzymes
Enzyme - a biological catalyst, composed of protein.
characteristics
increase rate of reaction
does not effect thermodynamics (can catalyze both exergonic and endergonic
reactions)
highly specific
not used up - recycled
Enzyme function substrate attaches to enzyme at active site
reaction takes place
enzyme is released from product
Mechanisms
1. alters geometry of substrate molecule slightly to enable reaction to proceed
2. holds substrates in proper orientation
3. concentrate substrates from dilute surroundings
Nomenclature
name ends in ase
usually descriptive of the type of reaction and substrate
e.g. substrate oxidase - enzyme that oxidizes substrate
substrate hydroxylase - enzyme that hydroxylates substrate
some names you just have to remember
amylase - starch degrading enzyme

2
cellulase - cellulase degrading enzyme
Some enzymes contain additional non-protein groups e.g. heme (cytochromes)
Coenzymes - bound loosely to the enzymes
may associate with other enzymes
serve as intermediate carriers of small molecules from one enzyme to
another
Enzyme Diversity
Microbial populations have the job of being the scavengers of the world. These
organisms (fungi and bacteria) contain a breadth of enzymes capable of degrading any
natural compound. Not every bacteria can degrade every compound, but for every
compound that has evolved naturally, a population of bacteria will have also evolved
capable of degrading the compound. The evolution of enzymes is a study of extreme
interest to environmental engineers and microbiologists, as we are now asking the
bacteria to metabolize compounds that are man made and have not been in the
environment long enough (in some cases) for the organisms to become adapted to them.
The study of enzyme evolution is in its infancy but it has been shown that in most cases a
new enzyme will evolve from an old enzyme that performs the same reaction on a more
natural chemical. For example if you think a compound would best be degraded by a
specific type of reaction, then you should expose bacteria (or fungi) that can do the type
of reaction you want to the new compound and hopefully sooner or later they will be able
to use the new compound. This may take a long time, but in most cases it will work
eventually. Many are trying to make designer enzymes, ones in which genetic
engineering has been used to purposefully modify genes and create new enzymes. This
may show great promise, some day, but a lot more work must be done to make the new
enzymes stable in the cell. Organisms that are exposed to the compound over long
periods of time and adapt to the compounds (enrichment) are mush more stable in the
environment than genetically engineered microorganisms.
Many biological reactions, especially in metabolism, involve the transfer of
electrons therefore they can also be classed as redox reactions. This means that one
compound is being reduced and the other is being oxidized.
Redox Reactions
See Chapter 2 from Rittman and McCarty or Appendix 1 in Brock
Oxidation - loss of ee.g. Fe+2 Fe+3 + e- (half reaction)
Reduction gain of electron
e.g. 1/2 O2 + 2H+ + 2e- H2O (half reaction)
free electrons do not exist in nature so oxidation and reduction must be coupled.

3
Balancing reactions
1. Balance electrons
2. Balance charge
3. Balance numbers of atoms on both sides
e.g. Coupling nitrate reduction to glucose oxidation
NO3- 1/2 N2
C6H12O6 6 CO2
electrons
N from +5 0 5 e- are added
glucose c = 0 CO2 (c = +4 x 6 = 24) 24 e- are lost.
to balance multiply nitrate equation by 24/5 or 4.8
4.8 NO3- + C6H12O6 2.4 N2 + 6CO2
Charges are not balanced (use H+ to balance - charge or put H+ on the other side to bal +)
4.8 NO3- + 4.8 H+ + C6H12O6 2.4 N2 + 6CO2
Number of atoms is not balanced
Use H2O to balance Os
Check
charge
C
N
O
H

4.8 NO3- + 4.8 H+ + C6H12O6 2.4 N2 + 6CO2 + 8.4 H2O

0
6
4.8
20.4
16.8

0
6
4.8
20.4
16.8

Redox Potential
tendency of substances to accept electrons and become reduced
written as reductions in table i.e. electrons on left
Eo - reduction potential in volts at pH 7
Eo > 0 reaction favorable
e.g. oxidize sulfide to sulfate with O2
4H2O + HS- SO4-2 + 8e- + 9H+

Eo = 0.217v

4
4H+ + O2 + 4e- 2H2O
balance eoverall
2O2 + HS- SO4-2 + H+

Eo = 0.82v

Eo = 1.037v
favourable

can convert to G
G = -n F Eo (where F is Faradays constant 96.48 kJ/volt; n is number of electrons)
= -8 (96.48) (1.037)
= -800 kJ/mol HSor directly from free energy of formation Gf (kJ/mol)
2 O2 + HS- SO4-2 + H+
0

12.05

-744.6

-39.83

G = -796 kcal/mol HSIn order for a cell to gain energy from a reaction, the cell must get the compound
into its interior so it can couple the chemical reaction to energy generation processes. As
we have discussed previously, the membrane is a barrier to the entry of large molecules
into the cell.

5
TRANSPORT
Most gasses and some small molecules can pass through the membrane but molecules
larger than glycerol must be transported across the cell membrane. The outer membrane
of Gram negative organisms contains embedded proteins called porins that allow most
monomeric, and some dimeric compounds to pass through.
A. Polymeric molecules
Polymeric molecules such as proteins, cellulose and starch are too big to enter
into the cell. So instead the cell exports enzymes that can break down the larger
molecules into smaller units that can be transported into the cell.
These enzymes are called extracellular enzymes and fall into two general
categories.
1. Hydrolases - these enzymes add water across a bond (the opposite to the
condensation that produced the polymers) thus cleaving the bond and producing
two new molecules. e.g.
protease - enzyme that cleaves the peptide bond of proteins resulting in the
release of amino acids.
cellulase - enzyme that cleaves cellulose into glucose monomers
amylase - enzyme that cleaves starch into glucose monomers
chitinase - enzyme that degrades chitin
2. Peroxidases - these enzymes introduce a free radical into a molecule thus
causing it to be unstable. The compound usually undergoes some kind of bond
cleavage as it becomes stable again. The enzymes require H2O2 (peroxide) for
their activity. The enzymes are most often produced by fungi that grow on
lignin (the woody material from plants) e.g. Phanerochaete chrysosporium.
The enzymes are not very specific in activity and can act on a wide variety of
substrates (usually aromatic)
These enzymes act outside the cell so they are not directly coupled to energy generation.
The monomer products of the enzyme activity are then brought inside the cell where their
metabolism can be coupled to energy generation.
There are several different mechanisms in which a compound can enter into a cell. Some
are more efficient than others and some require energy while others do not. The
compounds are all brought into the cell by way of special transport proteins imbedded in
the cell membrane. These proteins are very specific for specific compounds or groups of
compounds.

6
Transport Mechanisms
1. Diffusion
The compounds simply diffuse through the membrane, this relies on the
permeability of the compound and a concentration gradient. Compounds always diffuse
from a high concentration to a low concentration. The more difference in concentration
the faster the diffusion occurs. This requires no energy and is only applicable to gasses
and some very small molecules. There are some diffusion coefficients in your text
(transport section).
2. Facilitated diffusion
specific proteins allow some of the larger compounds in
relies on a concentration gradient
important in outer membrane porins
3. Active transport
Mechanisms by which the compounds are actively pumped across a membrane.
All require energy of some sort
All require specific protein carriers
All are saturable
Before we go on with this you must learn one more thing about the membrane. The
membrane is not permeable to protons or hydroxyl radicals. During normal metabolism
the cell pumps all protons out and keeps all hydroxyl ions in. What this does is sets up a
battery type situation with protons and other positive ions on the outside and none on the
inside. This creates a gradient of protons that make up a theoretical force that is used for
work when a proton is allowed into the cell. The force has been called the proton
motive force or PMF. It is composed of two separate smaller forces
pH = the proton gradient
= the charge gradient
Pictorial representation of PMF

7
This force is used for many things including transport.
Types of active transport (simple ones)
a) Symport
two compounds entering or leaving together
usually relies on the PMF
H+ moves inside the cell therefore creating enough energy to allow
the other compound to be pulled in with it.
We say that this is at the expense of pH
b) Antiport
one compounds comes in, the other goes out
energy usually from pH again
c) Uniport
single compound entering or leaving cell
usually only ions (+ can go in at expense of , - out)
More complicated Active Transport
d) Group translocation
substance being transported is altered as it crosses the membrane
example is the phosphoenolpyruvate dependent phosphotransferase
system. (you should all be familiar with this be now) Basically
the sugar is phosphorylated as it crosses the membrane. There are
several proteins involved. The phosphate is transferred from
phosphoenolpyruvate to glucose through enzyme intermediaries.
Text has an in depth explanation.
e) Binding Protein mediated transport
This is found in Gram negative cells only.
The Binding proteins (BPs) are located in the periplasm (space
between the two membranes)
The proteins bind the compound and deliver it to the membrane (a
conformational change upon binding causes the protein to become
hydrophobic and go towards the membrane.
Transport proteins in the membrane attract the BPs (hydrophobic
interactions again)
This keeps the concentration of the compound in the periplasm low
and allows the diffusion gradient across the outer membrane to
remain strong.
The protein undergoes another conformational change when it gets
to the transport protein and the compound is released to the
transport protein. The BP goes back out for more.
The transport protein uses a high level ~P bond and transports the
compound into the cell. The transport protein goes back to its
original conformation and is ready to accept another compound.

8
Microbial Energetics
We have just discussed some specific ways in which the cell couples energy
generating reactions to perform work that needs energy. These reactions require specific
proteins and use a small number of compounds that can carry energy from one part of the
cell to another.
1. Freely Diffusable Electron Carriers
These molecules diffuse throughout the cell and participate in all oxidation
reduction reactions either as electron acceptors or donors.
i) NAD - nicotinamide adenine dinucleotide
ii) NADP - NAD phosphate
These molecules are also proton carriers
or

NADH + H+ NAD+ + 2e- + 2H+

NADPH + H+ NADP+ + 2e- + 2H+

These are most often involved in dehydrogenation reactions and carry electrons from the
metabolic reactions to the electron transport chain for energy generation.
The two molecules can be interconverted by an enzyme but usually take part in different
types of reactions.
NAD - usually carries electrons to electron transport
NADP - usually carries electrons to biosynthetic pathways
2. Membrane Bound Carriers
These are compounds that are bound in the membrane and are part of the electron
transport chain. They accept electrons from the soluble carriers and pass them along
from one to another until the electron is passed on to the terminal electron acceptor.
You will get more on this later.
3. High Energy Phosphate Carriers
ATP
The best example is ATP. This molecule is the prime energy carrier of the cell. I
like to call it the money of the cell. Cleavage of ATP to ADP generates energy and can
be coupled to many reactions that require energy. ATP is not the highest energy carrier
but it has a middle range so that it is not too hard to make. The P bond can be transferred
from any one of the compounds above ATP to ATP and on down.
ATP Production
I - substrate level phosphorylation (SLP)

9
transfer of a high energy phosphate bond from an organic compound to ADP to
form ATP
e.g. These reactions form part of the pathway for the metabolism of glucose. In
the sequence an inorganic phosphate molecule is incorporated into an organic
molecule and then transferred to ADP to form ATP
glyceraldehyde-3-P + Pi + NAD NADH + 1,3-diphosphoglycerate
1,3-diphosphoglycerate + ADP phosphoglycerate + ATP + NAD
II - Conservation of High Energy Bonds
In organisms that are fermentative in nature, there is very little energy
around. The organisms employ another type of high energy bond (CoA
bond) which can be converted to a P bond by specific enzymes.
e.g.
succinate thiokinase (another important enzyme we will learn about)
succinyl CoA + GDP + Pi Succinate + GTP + CoA
or acetyl kinase
acetyl CoA + Pi Acetyl P + CoA (+ADP) acetate + ATP + CoA.
The name kinase suggests that the enzymes are involved in ATP synthesis.
Most organisms are capable of carrying out these types of reactions and they are the
major source of ATP in cells grown anaerobically in the dark but they do not contribute
much ATP in cells that are grown aerobically of under photosynthetic conditions.
III Oxidative phosphorylation
ATP formed when protons are allowed into the cell through a membrane bound
enzyme called F1Fo ATPase. (see Brock). F0 is a transmembrane protein and F1 is the
ATPase enzyme that couples the energy from the proton translocation to ATP production.
This uses pH of the PMF and is the major way of producing ATP for aerobically
growing cells.
IV Photophosporylation
ATP formed from light reactions in bacteria and plants. The ATP is produced in
the same way as for oxidative phosphorylation except the proton gradient is generated
differently.