Hogg S. Essential microbiology

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118 MICROBIAL METABOLISM

Figure 6.11 Competitive inhibition. In the presence of a competitive inhibitor (lower curve)

max

is reached eventually, but the apparent value of K

is increased. Since there are fewer

enzyme molecules in circulation, the apparent afﬁnity for its substrate is diminished

The competitive inhibitor is able to act in this way because its molecular structure is

sufﬁciently similar to that of the substrate for it to be able to ﬁt into the active site. The

effect is competitive because it depends on the relative concentrations of the substrate

and inhibitor. If the inhibitor is only present at a low concentration, its effect will be

minimal, since the number of enzyme–inhibitor interactions will be greatly outweighed

by reactions with the ‘proper’ substrate. The V

max

value for the enzyme is not reduced,

but it is only reached more gradually. The apparent afﬁnity of the enzyme for its substrate

is decreased, reﬂected by an increase in the K

(Figure 6.11).

Not all inhibitors act by competing for the active site, however. Non-competitive

inhibitors act by binding to a different part of the enzyme and in so doing alter its

three-dimensional conﬁguration (Figure 6.10b). Although they do not affect substrate

binding, they do reduce the rate at which product is formed. V

max

cannot be reached;

however, the value of K

, is unchanged (Figure 6.12). Such inhibitors may bind to either

the enzyme–substrate complex or to free enzyme. Both competitive and non-competitive

forms of inhibition are reversible, since the inhibitor molecule is relatively weakly bound

and can be displaced.

Irreversible inhibition, on the other hand, is due to the formation of a strong covalent

linkage between the inhibitor and an amino acid residue on the enzyme. As a result of its

binding, the inhibitor effectively makes a certain percentage of the enzyme population

permanently unavailable to catalyse substrate conversion.

Principles of energy generation

In this section, we shall consider how enzyme-catalysed reactions are involved in the

cellular capture and utilisation of energy.

Energy taken up by the cell, be it in the form of nutrients or sunlight, must be

converted into a usable form. A simple analogy is selling goods for cash, which you

can then use to buy exactly what you want. The ‘cash’ of cellular metabolism is a

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PRINCIPLES OF ENERGY GENERATION 119

Figure 6.12 Non-competitive inhibition. Since product is not formed as efﬁciently in the

presence of a non-competitive inhibitor (lower curve), V

max

is not reached. K

is not altered

compound called adenosine triphosphate (ATP). ATP is by far the most important of a

class of compounds known as high-energy transfer compounds, which store the energy

from the breakdown of nutrients (or trapped by photosynthetic pigments) and release

it when required by the cell. In catabolic reactions, in which molecules such as glucose

are broken down, energy is released in the form of ATP, which can then be utilised in

anabolic (synthetic) reactions.

ATP has a structure very similar to the nucleotides found in RNA, except it has

two additional phosphate groups (Figure 6.13). The bond that links the third phos-

phate group requires a lot of energy for its formation, and is often referred to as a

‘high-energy’ phosphate bond. Importantly for the cell, when this bond is broken, the

same large amount of energy is released, so when ATP is broken down to ADP and

a free phosphate group, energy is made available to the cell. It should be noted that

‘high-energy’ refers to the amount of energy needed to make or break the bond, and

not to any intrinsic property of the bond itself. The process of adding or removing a

phosphate group is called phosphorylation or dephosphorylation, respectively.

Oxidation--reduction reactions

Many metabolic reactions involve the transfer of electrons from one molecule to another;

these are called oxidation-reduction or redox reactions. When a molecule (or atom or

ion) loses an electron, it is said to be oxidized. (Note, that despite the terminology,

oxygen does not necessarily take part in the reaction.) Conversely, when an electron is

gained, the recipient is reduced (Figure 6.14).

Many metabolic reactions involve the loss of a hydrogen atom; since this contains one

proton and one electron, the reaction is regarded as an oxidation, because an electron

Not all of the energy is converted into ATP. A proportion of it is lost as heat, some of which allows the

enzyme-mediated reactions to proceed at a faster rate.

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120 MICROBIAL METABOLISM

Figure 6.13 Adenosine triphosphate (ATP). ATP is a nucleotide such as similar to those

depicted in Figures 2.20 and 2.21. Note the extra phosphate groups

has been lost:

C COO

–

C COO

–

+ 2H

+2e

–

Lactate Pyruvate

The lactate in the example above, by losing two hydrogen atoms, has automatically

lost two electrons and thus become oxidised to pyruvate. Oxidation reactions are

Reduction

electron

Oxidation

Figure 6.14 Oxidation–reduction reactions. When one molecule is oxidised, another is

simultaneously reduced. In the example shown, ‘X’ loses an electron and thus becomes

oxidised. By receiving the electron, ‘Y’ becomes reduced

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PRINCIPLES OF ENERGY GENERATION 121

Box 6.1 Coupled reactions

Many reactions in metabolic pathways, including glycolysis, can only take place if

they are coupled to a secondary reaction. The oxidising power necessary for the

conversion of glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate in step 6 of

glycolysis for example, is provided by the coenzyme NAD

. In the coupled reaction,

this becomes oxidised to NADH:

NAD

NADH

Glyceraldehyde-3-phosphate 1, 3-diphosphoglycerate

always associated with the transfer of energy from the oxidised substance to the reduced

substance.

Two important molecules that we shall encounter a number of times in the following

pages are the coenzymes nicotinamide adenine dinucleotide and nicotinamide adenine

dinucleotide phosphate; you will be relieved to learn that they are nearly always referred

to by their abbreviations, NAD

and NADP

, respectively! Both are derivatives of the

B vitamin niacin, and each can exist in an oxidised and a reduced form:

NAD

+ H

+ 2e

–

NADH

NADP

+ H

+ 2e

–

NADPH

Oxidised Reduced

They are to be found associated with redox reactions (see Box 6.1), acting as carrier

molecules for the transfer of electrons. In the oxidation of lactate shown above, the

oxidising power is provided by the reduction of NAD

, so the full story would be:

Lactate Pyruvate

NAD

NADH + H

As the lactate is oxidised, so the NAD

in the coupled reaction is reduced. It is said to

act as the electron acceptor. NAD

/NADH is generally involved in catabolic reactions,

and NADP

/NADPH in anabolic ones.

As the equation above shows, there can be no oxidation without reduction, and vice

versa; the two are irrevocably linked. The tendency of an compound to lose or gain

electrons is termed its redox potential (E

) (Box 6.2).

In the following section, we shall examine chemoheterotrophic metabolism, used by

the majority of microorganisms to derive cellular energy from the oxidation of carbo-

hydrates. Other groups have evolved their own systems of energy capture, and these

will be considered later in the chapter.

Figure 6.15 provides a summary of the catabolic (breakdown) pathways used

by heterotrophs. Complex nutrients such as proteins and polysaccharides must be

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122 MICROBIAL METABOLISM

Box 6.2 Redox potentials

Substances vary in the affinity they have for binding electrons; this can be measured

as their oxidation--reduction potential or redox potential, relative to that hydrogen.

The flow of electrons in the electron transport chain (see Fig. 6.21) occurs because

the carriers are arranged in order of their redox potentials, with each having a

greater electron affinity (more positive redox potential) than its predecessor. Thus

electrons are donated to carriers with a more positive redox potential.

−

Strongly negative redox potential

(Good electron donors)

Reduction

Potential

+ Strongly positive redox potential

(Good electron acceptors)

enzymatically broken down and converted to substances that can then enter one of the

degradative pathways that lead to energy production.

Glucose is the carbohydrate most widely used as an energy source by cells, and the

processes by which it is broken down in the presence of oxygen to give carbon dioxide

and water are common to many organisms. These have been very thoroughly studied

and can be summarised:

+ 6O

−→ 6CO

+ 6H

Figure 6.15 Catabolic pathways in heterotrophs. Pathways for the catabolism of proteins,

nucleic acids and lipids as well as carbohydrates can all feed into the tricarboxylic acid cycle

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PRINCIPLES OF ENERGY GENERATION 123

Figure 6.16 Glycolysis. Two molecules of ATP are ‘spent’ in the ﬁrst stage of glycolysis, in

which glucose is converted into the 3-carbon compounds glyceraldehyde 3-phosphate and

dihydroxyacetone phosphate. During the second stage, four ATPs are produced per molecule

of glucose, so there is a net gain of two ATPs. In addition, reducing power is generated in the

form of NADH (two molecules per molecule of glucose). See Figure 6.17 for a more detailed

depiction of the reactions involved in glycolysis

What this equation, crucially, does not show is that as a result of this process, energy is

released, and stored in the form of 38 molecules of ATP, so for completeness, we need

to add to the respective sides:

38ADP + 38Pi −→ 38ATP

(Pi = inorganic phosphate group)

The release of the energy contained within a molecule of glucose does not occur in a sin-

gle reaction, but happens gradually, as the result of numerous reactions linked together

in biochemical pathways, the ﬁrst of which is glycolysis (Figure 6.16). Glycolysis can

occur with or without oxygen, and is common to both aerobic and anaerobic organ-

isms. Oxygen is essential, however, for aerobic respiration, by which ATP is generated

from the products of glycolysis. Anaerobes proceed down their own pathways follow-

ing glycolysis as we shall see, but these lack the ATP-generating power of the aerobic

process.

Why glucose?

By concentrating on glucose catabolism in this way, you may think we are ignoring the

fate of other nutrient molecules. If you take another look at Figure 6.15, however, you

will notice that the breakdown products of lipids, proteins and nucleic acids also ﬁnd

their way into our pathway sooner or later, having undergone transformations of their

own.

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124 MICROBIAL METABOLISM

Glycolysis

The initial sequence of reactions, in which a molecule of glucose is converted to two

molecules of pyruvate

∗

, is called glycolysis (Figures 6.16 and 6.17). In the ﬁrst phase

of glycolysis, glucose is phosphorylated and its six-carbon ring structure rearranged,

before being cleaved into two three-carbon molecules. In the second phase, each of

these undergoes oxidation, resulting in pyruvate.

Also known as the Embden–Meyerhof pathway, glycolysis is used for the metabolism

of simple sugars not just by microorganisms, but by most living cells. The pathway,

which takes place in the cytoplasm, comprises a series of 10 linked reactions, in which

each molecule of the six-carbon glucose is converted to two molecules of the three-

carbon pyruvate, with a net gain of two molecules of ATP.

The full pathway of reactions is shown in Figure 6.17. Note how, in terms of energy,

glycolysis can be divided conveniently into a ‘sowing’ phase, in which two molecules of

ATP are expended per molecule of glucose, followed by a ‘reaping’ phase which yields

four molecules of ATP. The overall energy balance is therefore a gain of two molecules

of ATP for each molecule of glucose oxidised to pyruvate. In addition, the second phase

features the conversion of two molecules of NAD

to NADH, which, as we will see,

act as an important source of reducing power in subsequent pathways.

The reactions by which ATP is generated from ADP in the second phase of glycolysis

are examples of substrate-level phosphorylation, so-called because the phosphate group

is transferred directly from a donor molecule.

What happens next to the pyruvate produced by glycolysis depends on the organism

concerned, and on whether the environment is aerobic or anaerobic; we shall look at

these possibilities in due course.

Glycolysis is not the only way to metabolise glucose

Although glycolysis is widespread in both the microbial and nonmicrobial worlds, sev-

eral bacterial types use alternative pathways to oxidise glucose. For certain Gram-

negative groups, notably the pseudomonads (see Chapter 7), the main route used is

the Entner–Doudoroff pathway, producing a mixture of pyruvate and glyceraldehyde-

3-phosphate (Figure 6.18). The former, like that produced in glycolysis, can enter a

number of pathways, while the latter can feed into the later stages of glycolysis. The

net result of catabolism by the Entner–Doudoroff pathway is the production of one

molecule each of ATP, NADH and NADPH per molecule of glucose degraded.

A secondary pathway, which may operate in tandem with glycolysis or the Entner–

Doudoroff pathway, is the pentose phosphate pathway, sometimes known as the hexose

monophosphate shunt (Figure 6.19). Like glycolysis, the pathway can operate in the

presence or absence of oxygen. Although glyceraldehyde-3-phosphate can once again

enter the glycolytic pathway and lead to ATP generation, for most organisms the path-

way has a mainly anabolic (biosynthetic) function, acting as a source of precursor

molecules for other metabolic pathways. The pentose phosphate pathway is a useful

∗

At physiological pH, carboxylic acids such as pyruvic acid and citric acid are found in their ionised form

(pyruvate, citrate); however long-established traditions persist, and you may well ﬁnd reference elsewhere to

the ‘-ic acid’ forms.

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PRINCIPLES OF ENERGY GENERATION 125

Glucose

ATP

hexokinase

ADP

Glucose 6-phosphate

phosphohexose

isomerase

Fructose 6-phosphate

ATP

phosphofructokinase

ADP

Fructose 1,6-bisphosphate

Glyceraldehyde Dihydroxyacetone

3-phosphate phosphate

triose phosphate

isomerase

NAD

glyceraldehyde 3-phosphate

NADH dehydrogenase

1,3-Bisphosphoglycerate

ADP

phosphoglycerate

ATP

kinase

3-Phosphoglycerate

phosphoglycerate

mutase

x 2

2-Phosphoglycerate

enolase

Phosphoenolpyruvate

ADP

pyruvate kinase

ATP

Pyruvate

Figure 6.17 Glycolysis: a more detailed look. Glycolysis comprises 10 separate enzyme-

catalysed reactions. Of the two 3-carbon compounds formed in the ﬁrst stage, dihydroxyace-

tone phosphate cannot directly enter the later part of the pathway, but must ﬁrst be converted

to its isomer glyceraldehyde-3-phosphate. For each molecule of glucose, two molecules of

each compound are therefore produced from this point onwards, and the yield of ATP and

NADH is likewise doubled

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126 MICROBIAL METABOLISM

Glucose

ATP

ADP

Glucose 6-phosphate

NADP

NADPH

6-Phosphogluconate

2-keto 3-deoxy-6-phosphogluconate

Glyceraldehyde

Pyruvate

3-phosphate

NAD

NADH

ADP

ATP

Steps 6–10 of glycolysis (x 2)

ADP

ATP

Pyruvate

Figure 6.18 The Entner–Doudoroff pathway: an alternative way to metabolise glucose.

The products of the pathway are pyruvate and glyceraldeyde-3-phosphate (G3-P). There is

a loss of one molecule of ATP in the opening reaction, however when the G3-P joins the

later stages of glycolysis, two ATPs are generated, giving a net balance of +1. In addition,

the pathway yields one molecule each of NADH and NADPH. The intermediate compound

6-phosphogluconate can enter the pentose phosphate pathway and be decarboxylated to the

5-carbon compound ribulose-5-phosphate (see Figure 6.19)

source of reducing power in the form of NADPH. In addition it acts as an important

source of precursors in the synthesis of essential molecules; ribose-5-phosphate is an

important precursor in the synthesis of nucleotides, while the four-carbon erythrose

4-phosphate is required for the synthesis of certain amino acids and ribulose-5-

phosphate is an intermediate in the Calvin cycle of carbon ﬁxation (see later in this

chapter).

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Figure 6.19 The pentose phosphate pathway. Operating simultaneously with glycolysis,

the pathway serves as a source of precursors for other metabolic pathways. The metabolic

fate of intermediates is indicated in italics. Circled numbers next to each molecule denote

the number of carbons

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