Review
The Limits of Enzyme Specificity and the Evolution of Metabolism

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Highlights

Substrate specificity cannot be absolute and is inherently limited.

The maximum capacity to discriminate between alternative substrates can be relatively low, and in any case it is seldom approached owing to evolutionary constraints.

In some enzymes of primary metabolism, substrate promiscuity is favored, although this may interfere with high flux and efficient regulation.

In other cases, enzymes acting on alternative substrates generate toxic or useless products; however, these can be destroyed or recycled by repair enzymes.

The limited substrate specificity of enzymes often results in the production of non-standard metabolites which contribute to the complexity of the metabolome.

Substrate promiscuity helps to fuel an ‘underground’ network of reactions which may represent a basis for further evolution and diversification of metabolism.

The substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons – for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic ‘messiness’, from which both liabilities and opportunities ensue in the evolution of metabolic systems.

Section snippets

Many Metabolic Enzymes Are Not Strictly Substrate-Specific

It is now well appreciated that a substantial fraction of metabolic enzymes can catalyze reactions of different types and/or with different substrates 1, 2. The former behavior is termed catalytic promiscuity [3], the latter is usually called substrate promiscuity [4] (because these terms may be equivocal, Box 1 explains the definition of ‘promiscuity’ used here and compares it to a stricter definition accepted by evolutionary biochemists). Although this paper is essentially concerned with

Substrate Specificity, Discrimination, and Binding Energy

In contrast to catalytic efficiency, which can be gauged in reference to an absolute scale of ‘catalytic perfection’ 10, 11, specificity is a relative concept because it requires comparison between given alternative substrates. In fact, specificity is formally defined as the ability of an enzyme to discriminate between two potential substrates, in the presence of both compounds 12, 13. In a biological context, specificity entails acting on a single substrate in preference to a multitude of

Theoretical and Empirical Limits of Substrate Specificity

That substrate specificity is inherently limited was first glimpsed by Pauling 60 years ago [15] in relation to the process of aminoacyl-tRNA synthesis. He focused in particular on the case of isoleucyl-tRNA synthetase that must distinguish between isoleucine and valine. The two amino acids differ only by one methyl group, and the difference in binding energy between them could therefore not be greater than the energy provided by the terminal CH3 group of L-Ile. This has been estimated to be

Detrimental Activities with Alternative Substrates May Be Redressed by Repair Enzymes

An opposite scenario arises when the activity of a metabolic enzyme with an alternative substrate is seriously detrimental to fitness. In fact, every such reaction may generate a product that is useless for the cell (a metabolic dead-end, implying a waste of resources) or that is even toxic [44], and this is expected to elicit strong evolutionary pressure to improve the specificity of the enzyme. However, as discussed, there are limits to such an improvement. Preventing access of the enzyme to

When Does Activity with Alternative Substrates Depend on Neutral Drift?

In a final scenario, the alternative reaction catalyzed by an enzyme might have no significant (positive or negative) effects on the system fitness, and thus it would be invisible to natural selection and essentially subject to neutral drift. This is often assumed as the ‘default’ case [11], but positive proof is scarce. We have seen above that some reactions which, based on the discrimination factor, would appear very negligible, do become liabilities because the promiscuous enzyme and the

Substrate Promiscuity Contributes to Underground Metabolism

As seen in the sections above, enzymes have an unavoidable tendency to act on alternative, available substrates. Even when (perhaps not very often) this tendency is restrained to near the minimum level allowed by chemistry, and even in the presence of ‘repair’ systems, these enzymes will generate alternative products, many of which will be nonstandard metabolites. Substrate promiscuity must be hence considered as a major contribution to the complexity of the metabolome – together with catalytic

Concluding Remarks

The results and arguments reviewed here show that, although perfect substrate specificity is essentially unattainable, metabolic enzymes are often much less selective than they could be. Furthermore, enzyme activities with alternative substrates are subject to distinct selective pressures. They can be fostered by natural selection until they reach levels that are most useful for fitness, or repressed to levels at which they are no longer harmful. When deleterious side-reactions cannot be

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