Turnover reactions, enzyme

Figure 1.9 Reaction cycle for an aspartyl protease illustrating the conformational changes within the active site that attend enzyme turnover. Source Model based on experimental data summarized in Northrop (2001). 

The examples above serve to illustrate that the conformational dynamics of enzyme turnover create multiple, specific binding pocket configurations throughout the reaction pathway, each representing a distinct opportunity for drug binding and inhibition. 

In addition to KM and vmax, the turnover number (molar activity) and the specific activity are two important parameters in enzyme catalyzed reactions. The turnover number indicates the number of substrate molecules converted per unit time by a single enzyme molecule. The specific activity is given in units and one international unit (i.u.) is the amount of enzyme that consumes or forms 1 pmol of substrate or 1 pmol of product per minute under standard conditions. 

As in the luminol case, the main role of the enhancer (EnH) seems to be related to turnover of the enzyme, generating enhancer radicals (En rad) in the process that are capable of oxidizing the acridan ester (AcH). The structure of the enhancer obviously is very important. To accelerate HRP turnover, the enhancer must on the one hand be able to rapidly react with the reactive HRP intermediates Cl and especially CII (k2 and k3 large). On the other hand, the oxidized enhancer intermediate (radical or radical cation) must be able to oxidize the acridan ester (light-generating step). This last reaction also depends on the structure of the acridan ester in a very unfavorable case, adding an enhancer for enzyme turnover could actually diminish the light production if k 4 > fct (Fig. 5), i.e., if the enhancer radical would not be able to oxidize the acridan ester. 

An outline mechanism for tyrosine activation has been proposed (Fersht, 1975 Fersht et al., 1975a,b Ward and Fersht, 1988a) on the basis of conventional kinetic and binding studies, and this is shown in (49). For the aminoacylation step, some aspects of the reaction are still not known such as the point at which AMP is displaced, but the currently preferred mechanism (Fersht and Jakes, 1975 Ward and Fersht, 1988b) is that given in (50). This is compatible with the observed kinetics which show that two moles of tyrosine bind in each enzyme turnover during which one molecule of Tyr-tRNA appears. 

N-Carbobenzoxy-L-alanine-/>-nitrophenyl ester is a specific substrate for elastase in which the rate-limiting step is deacylation, that is, hydrolysis of the acyl-enzyme intermediate. In 70% methanol over a reasonable temperature range the energy of activation of the turnover reaction, that is, deacylation, is 15.4 kcal mol. In the pH 6-7 region in this cryoprotective solvent, the turnover reacdon can be made negligibly slow at temperatures of -50 C or below. Under such conditions/i-nitro-phenol is released concurrent to acyl enzyme formation in a 1 1 stoichiometry with active enzyme in the presence of excess substrate. In other words, even at low temperatures, the acylation rate is much faster than deacylation and the acyl enzyme will accumulate on the enzyme. The rate of acyl-enzyme formation can be monitored by following the rate of p-nitrophenol release, and thus the concentration of trapped acyl enzyme may be determined. This calculadon has been carried out and... 

The turnover reaction of hydrolysis of 2, 3 -CMP could be made negligibly slow at temperatures below -60°C at pH 3-6 in 70% methanol, and below -35°C at pH 2.1. The rate of the catalytic reaction using crystalline enzyme was found to be 50-fold slower than that of dissolved enzyme for cyclic phosphate hydrolysis, and 200-fold slower for dinucleotide hydrolysis (presumably the greater reduction for the larger substrate reflects increased diffusional hindrance by the small solvent chan-... 

In the absence of an enzyme, the reaction rate v is proportional to the concentration of substance A (top). The constant k is the rate constant of the uncatalyzed reaction. Like all catalysts, the enzyme E (total concentration [E]t) creates a new reaction pathway, initially, A is bound to E (partial reaction 1, left), if this reaction is in chemical equilibrium, then with the help of the law of mass action—and taking into account the fact that [E]t = [E] + [EA]—one can express the concentration [EA] of the enzyme-substrate complex as a function of [A] (left). The Michaelis constant lknow that kcat > k—in other words, enzyme-bound substrate reacts to B much faster than A alone (partial reaction 2, right), kcat. the enzyme s turnover number, corresponds to the number of substrate molecules converted by one enzyme molecule per second. Like the conversion A B, the formation of B from EA is a first-order reaction—i. e., V = k [EA] applies. When this equation is combined with the expression already derived for EA, the result is the Michaelis-Menten equation. 

The ratio of the turnover number (i.e., Emax/[Etotai]) to the Xn, value of a substrate in a particular enzyme-catalyzed reaction. When kcat and are the true steady-state parameters, this ratio (or the ratio Emax/T m) is an excellent gauge of the specificity of the enzyme for that substrate. The larger the ratio, the more effective that substrate is used by the enzyme under study. In addition, the effects of a number of mechanistic probes of enzyme action on this ratio (for example, pH effects, isotope effects, temperature effects, the influence of various modifiers, etc.) can provide much information on the catalytic and binding mechanism. See... 

Km and Umax have different meanings for different enzymes. The limiting rate of an enzyme-catalyzed reaction at saturation is described by the constant kcat, the turnover number. The ratio kcat/Km provides a good measure of catalytic efficiency. The Michaelis-Menten equation is also applicable to bisubstrate reactions, which occur by ternary-complex or Ping-Pong (double-displacement) pathways. 

FIGURE 6-19 Pre-steady state kinetic evidence for an acyl-enzyme intermediate. The hydrolysis of p-nitrophenylacetate by chymotrypsin is measured by release of p-nitrophenoi (a colored product). Initially, the reaction releases a rapid burst of p-nitrophenol nearly stoichiometric with the amount of enzyme present. This reflects the fast acylation phase of the reaction. The subsequent rate is slower, because enzyme turnover is limited by the rate of the slower deacylation phase. 

Most enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 10 times faster than uncatalyzed reactions. Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into product each second. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number. 

It was once thought that the rate of equilibrium of the catalytic acid and basic groups on an enzyme with the solvent limited the rates of acid- and base-catalyzed reactions to turnover numbers of 103 s 1 or less. This is because the rate constants for the transfer of a proton from the imidazolium ion to water and from water to imidazole are about 2 X 103 s 1. However, protons are transferred between imidazole or imidazolium ion and buffer species in solution with rate constants that are many times higher than this. For example, the rate constants with ATP, which has a pKa similar to imidazole s, are about I0 J s 1 M-1, and the ATP concentration is about 2 mM in the cell. Similarly, several other metabolites that are present at millimolar concentrations have acidic and basic groups that allow catalytic groups on an enzyme to equilibrate with the solvent at 107 to 108 s-1 or faster. Enzyme turnover numbers are usually considerably lower than this, in the range of 10 to 103 s-1, although carbonic anhydrase and catalase have turnover numbers of 106 and 4 X 107 s 1, respectively. 

Similar experiments in the back reaction of hexokinase have shown that glucose-6-P dissociates from E-glucose-6-P about 160 times faster than the enzyme turnover in the reverse reaction, and very slowly from E-glucose-6-P-MgADP (66). 

As noted earlier, the velocity of any enzyme-catalyzed reaction is dependent upon the amount of effective enzyme present. Enzyme biosynthesis, like that of all proteins, is under genetic control, a long-term process. Biosynthesis of enzymes may be increased or decreased at the genome level. Various hormones can activate or repress the mechanisms controlling gene expression. Enzyme levels are the result of the balance between synthesis and degradation. This enzyme turnover may be altered by diverse physiological conditions, by hormone effects, and by the level of metabolites. 

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