Nonproductive binding

Poor substrates bind to the enzyme in a large number of different ways, only one of which is correct. Good substrates bind only in the proper way. 

The INDUCED-FIT model for enzyme specificity says that good substrates must be able to cause the enzyme to change shape (conformation) so that catalytic and functional groups on the enzyme are brought into just the right place to catalyze the reaction. Bad substrates are bad because they aren t able to make the specific interactions that cause the conformation change, and the enzyme stays in its inactive conformation. 

Good substrates bind to the enzyme in only the correct way 

The NONPRODUCTIVE BINDING model suggests that while good substrates bind in only one, correct way, bad substrates usually bind to the enzyme incorrectly and cannot react. 

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The reaction of cyclohexaamylose with a series of p-carboxyphenyl esters is an example of a decelerating effect which may be clearly attributed to nonproductive binding. Rate effects imposed by cyclohexaamylose on the hydrolyses of three such esters are summarized in Table IX. As the hydrophobicity of the ester function is increased by alkyl substitution, the hydrolysis is inhibited the stability of the inclusion complex, on the other... 

The result of equation 3.39 for nonproductive binding is quite general. It applies to cases in which intermediates occur on the reaction pathway as well as in the nonproductive modes. For example, in equation 3.19 for the action of chy-motrypsin on esters with accumulation of an acylenzyme, it is seen from the ratios of equations 3.21 and 3.22 that kQJKM = k2IKs. This relationship clearly breaks down for the Briggs-Haldane mechanism in which the enzyme-substrate complex is not in thermodynamic equilibrium with the free enzyme and substrates. It should be borne in mind that KM might be a complex function when there are several enzyme-bound intermediates in rapid equilibrium, as in equation 3.16. Here kcJKM is a function of all the bound species. 

Nonproductive binding. If the enzyme has binding modes for S other than the catalytically productive mode, these will favor [ES] in the equilibrium. 

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). 

Strain, induced fit, nonproductive binding, and steady state kinetics... 

Nonproductive binding. The larger substrate binds in the productive mode only, but the smaller one, in addition to binding more weakly in the productive mode, binds in nonproductive modes, lowering the KM. The fccat is correspondingly lower. 

Nitrocellulose filters 205 Non-Arrhenius kinetics 555, 598 Nonbonded interactions 325-332 Nondisruptive deletion 426, 560 Nonequilibrium dialysis 202 Nonproductive binding 114-118, 371-372... 

This is known as nonproductive binding. The effect of such binding on the Michaelis-Menten mechanism is. to lower both the kcat and the Ku. The kcu is lowered since, at saturation, only a fraction of the substrate is bound productively. The Km is lower than the Ks because the existence of additional binding modes must lead to apparently tighter binding. 

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. 

Nonproductive binding modes do not affect the pH dependence of kC JKM or MKm, for the reasons discussed in the previous case. 

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. 

Figure 12.8 Nonproductive binding with substrates and lysozyme. Small substrates may bind at alternative sites along the extended active site of lysozyme, avoiding the cleavage site, which has a lower affinity.

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. 

The enzyme uptake varied significantly according to the enzyme source, type, loading, and incubation time. The typical uptake for amylase ranged between 20 and 60%, whereas for cellulase, only 7-10% uptake was obtained. Since these uptake measurements simply reflect losses in soluble enzyme activity from the immobilization solution, they set a maximum on the activity that may be expressed by the immobilized enzyme. The actual activity is expected to be less, once losses resulting from shear, conformational changes, and nonproductive binding are taken into account. Direct measurements of immobilized enzyme activity therefore provide the best measure of the effectiveness of a particular immobilization technique. 

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