Specificity nonproductive binding

The most-studied enzyme in this context is chymotrypsin. Besides being well characterized in both its structure and its catalytic mechanism, it has the advantage of a very broad specificity. Substrates may be chosen to obey the simple Michaelis-Menten mechanism, to accumulate intermediates, to show nonproductive binding, and to exhibit Briggs-Haldane kinetics with a change of rate-determining step with pH. 

Specificity, in the sense of discrimination between competing substrates, is independent of the above three effects. The reasons are discussed in detail in Chapter 13. The basic reason is that specificity depends on kcal/KM, and strain and nonproductive binding do not affect the value of kcJKM because it is independent of interactions in the ES complex (equations 12.10 and 3.36). Equation 12.16 shows that induced fit does alter kcal/KM for the active conformation, but equally for all substrates (i.e., by a factor of K). 

The important conclusion is that specificity, in the sense of discrimination between two competing substrates, is determined by the ratios of kcJKM and not by Ku alone. Since km/KM is unaffected by nonproductive binding (section E) and by the accumulation of intermediates (section F), these phenomena do not affect specificity (see Chapter 13). Note that equation 3.44 holds at all concentrations of substrates. 

Although nonproductive binding is not a mechanism for increasing KM, it is appropriately discussed here since it gives rise to effects that are qualitatively similar to those of strain and induced fit. This theory was originally invoked to account for specificity in the relative reactivities of larger, specific substrates compared with smaller, nonspecific substrates. It is assumed that as well as the productive binding mode at the active site, there are alternative, nonproductive modes in which the smaller substrates may bind and not react. 

The difference between the two meanings is crucial to the status of strain, induced fit, and nonproductive binding in catalysis. As we discussed in Chapter 12 and as we shall amplify below, these do not affect biological specificity, since they alter kcat and KM in a mutually compensating manner without altering kcJKM. 

Nonproductive binding. It was shown in Chapter 3, section E, that nonproductive binding does not alter kcJKM but decreases both kCSLt and KM while maintaining their ratio. Specificity is unaffected. 

Studies on the substrate specificity of an enzyme aim to probe the structural requirements for efficient catalysis. In such studies, it is best to correlate the kinetic parameter kJK with substrate structure and not K, because the latter is often compromised by nonproductive binding (Fersht, 1985). For the PPIs, specificity studies have probed three general structural domains of simple peptide substrates (1) length of the substrate, (2) P/ residue (i.e.. Pro replacements), and, (3) Pj residue. 

Inhibitors act either reversibly or irreversibly. Reversible inhibitors include various modes of action competitive, noncompetitive, and uncompetitive [18]. A competitive inhibitor competes with the substrate for the active site of the enzyme and forms a nonproductive enzyme-inhibitor complex. A noncompetitive inhibitor binds to a site distinct from that which binds the substrate and, by changing the surface properties or conformation, inactivates the enzyme [19]. Alternatively, an uncompetitive inhibitor binds at a site distinct from the substrate but binds exclusively to an enzyme-substrate complex rather than to a free enzyme. In comparison, irreversible inhibitors are more specific, in that they must combine with or destroy a functional group on the enzyme that is essential for its activity. 

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