Environmental factors affect enzyme activity

The rate at which an enzyme converts its substrate into product is called its velocity (v), and is affected by a variety of factors.

Temperature

The rate of any chemical reaction increases with an increase in temperature due to the more rapid movement of molecules, and so it is with enzyme-catalysed reactions, until a peak is reached (the optimum temperature) after which the rate rapidly falls away. What

Figure 6.5 Disruption of an enzyme's three-dimensional structure causes denaturation. Disruption of the bonds that form the secondary and tertiary protein structure of an enzyme lead to a loss of catalytic activity, as the amino acids forming the active site are pulled apart. a) From Bolsover, SR , Hyams, JS, Jones, S, Shepherd, EA & White, HA: From Genes to Cells, John Wiley & Sons, 1997. Reproduced by permission of the publishers

Figure 6.5 Disruption of an enzyme's three-dimensional structure causes denaturation. Disruption of the bonds that form the secondary and tertiary protein structure of an enzyme lead to a loss of catalytic activity, as the amino acids forming the active site are pulled apart. a) From Bolsover, SR , Hyams, JS, Jones, S, Shepherd, EA & White, HA: From Genes to Cells, John Wiley & Sons, 1997. Reproduced by permission of the publishers causes this drop in the velocity? Recall from Chapter 2 that the very ordered secondary and tertiary structure of a protein molecule is due to the existence of numerous weak molecular bonds, such as hydrogen bonds. Disruption of these by excessive heat results in denaturation (Figure 6.5), that is, an unfolding of the three-dimensional structure. In the case of an enzyme, this leads to changes in the configuration of the active site, and a loss of catalytic properties. The effect of temperature on enzyme activity is shown in Figure 6.6. The graph can be thought of as a composite of two lines, one increasing with temperature due to the rise in thermal energy of the substrate molecules, and one falling due to denaturation of the protein structure. Before the optimum temperature it is the former which dominates, then the effect of the latter becomes more pronounced, and takes over completely.

Temperature (°C)

Figure 6.6 Effect of temperature on enzyme activity. Below the optimum temperature, the rate of reaction increases as the temperature rises. Above the optimum, there is a sharp falling off of reaction rate due to thermal denaturation of the enzyme's three-dimensional structure

Temperature (°C)

Figure 6.6 Effect of temperature on enzyme activity. Below the optimum temperature, the rate of reaction increases as the temperature rises. Above the optimum, there is a sharp falling off of reaction rate due to thermal denaturation of the enzyme's three-dimensional structure

Bases Denature Enzyme
Figure 6.7 Effect of pH on enzyme activity. Either side of the optimum pH value, changes in ionisation of amino acid side chains lead to protein denaturation and a loss of enzyme activity

Enzyme velocity is similarly affected by the prevailing pH. Once again, this is due to alterations in three-dimensional protein structure. Changes in the pH affect the ionisation of charged 'R'-groups on amino acids at the active site and elsewhere, causing changes in the enzyme's precise shape, and a reduction in catalytic properties. As with temperature, enzymes have an optimum value at which they operate most effectively; when the pH deviates appreciably from this in either direction, denaturation occurs, leading to a reduction of enzyme activity (Figure 6.7).

Microorganisms are able to operate in a variety of physicochemical environments, a fact reflected in the diversity of optimum values of temperature and pH encountered in their enzymes.

Substrate concentration

Under conditions where the active sites of an enzyme population are not saturated, an increase in substrate concentration will be reflected in a proportional rise in the rate of reaction. A point is reached, however, when the addition of further substrate has no effect on the rate (Figure 6.8). This is because all the active sites have been occupied and the enzymes are working flat out; this is called the maximum velocity (Vmax). A measure of the affinity an enzyme has for its substrate (i.e. how tightly it binds to it) is given by its Michaelis constant (Km). This is the substrate concentration at which the rate of reaction is half of the Vm Km are more easily determined experimentally by plotting the reciprocals of [S] and V to obtain a straight line (Figure 6.9).

The Michaelis-Menten equation relates the rate of a reaction to substrate concentration, [S]:

vmax[s]

Figure 6.8 Enzyme activity is influenced by substrate concentration. The initial rate of reaction (vo) is proportional to substrate concentration at low values of [S]. However, when the active sites of the enzyme molecules become saturated with substrate, a maximum rate of reaction (Vmax) is reached. This cannot be exceeded, no matter how much the value of [S] increases. The curve of the graph fits the Michaelis-Menten equation. Km is the value of [S] where v = ^p.

Figure 6.8 Enzyme activity is influenced by substrate concentration. The initial rate of reaction (vo) is proportional to substrate concentration at low values of [S]. However, when the active sites of the enzyme molecules become saturated with substrate, a maximum rate of reaction (Vmax) is reached. This cannot be exceeded, no matter how much the value of [S] increases. The curve of the graph fits the Michaelis-Menten equation. Km is the value of [S] where v = ^p.

1

/

V

/

■1 -1

/

[S] " Km

X

/ /

S 1

/

^max

Is

V

Figure 6.9 The Lineweaver-Burk plot. Plotting the reciprocal values of Vo and [S] enables values of Km and Vmax to be derived from the intercepts on a straight-line graph

Some enzymes do not obey Michaelis-Menten kinetics. The activity of allosteric enzymes is regulated by effector molecules which bind at a position separate from the active site. By doing so, they induce a conformational change in the active site that results in activation or inhibition of the enzyme. Thus effector molecules may be of two types, activators or inhibitors.

Enzyme inhibitors

Many substances are able to interfere with an enzyme's ability to catalyse a reaction. As we shall see in Chapter 14, enzyme inhibition forms the basis of several methods of microbial control, so a consideration of the main types of inhibitor is appropriate here.

Perhaps the easiest form of enzyme inhibition to understand is competitive inhibition. Here, the inhibitory substance competes with the normal substrate for access to the enzyme's active site; if the active site is occupied by a molecule of inhibitor, it can't bind a molecule of substrate, thus the reaction will proceed less quickly (Figure 6.10a).

Active site of enzyme

Competitive inhibitor

Products No Products b)

Substrate

Active site of enzyme

Non-competitive inhibitor

Products

Products

Reduced rate of product formation

Figure 6.10 Enzyme inhibition. (a) A competitive inhibitor mimics the structure of the normal substrate molecule, enabling it to fit into the active site of the enzyme. Although it is not acted on by the enzyme and no products are formed, such an inhibitor prevents the normal substrate gaining access to the active site. (b) A non-competitive substance binds to a second site on the enzyme and thus does not affect substrate binding; however distortion of the enzyme molecule makes catalysis less efficient

Figure 6.11 Competitive inhibition. In the presence of a competitive inhibitor (lower curve) Vmax is reached eventually, but the apparent value of Km 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 Vmax 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 Km (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. Vmax cannot be reached; however, the value of Km, 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.

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  • dawit
    Why is enzyme activity related to microbial growth?
    8 years ago

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