Enzymatics substrate affinity

The diagnosis of PK deficiency depends on the determination of quantitative enzyme activity or qualitative abnormalities of the enzyme. In 1979, the International Committee for Standardization in Haematology (ICSH) established methods for the biochemical characterization of red blood cell PK variants (M22). Since the establishment of these methods, many PK-deficient cases have been characterized, including 13 cases of homozygous PK deficiency. Residual red blood cell PK activity is not usually associated with phenotypic severity,whereas enzymatic characteristics such as decreased substrate affinity, thermal instability, or impaired response to the allosteric activator fructose-1,6-diphosphate (F-1,6-DP) correspond to a more severe phenotype. 

Equation 24" Is considered to be a cornerstone to the understanding of enzyme catalysis In terms of the magnitude of enzyme-substrate affinity In the transition state and how this affinity affects the energy of activation In favor of enzymatic catalysis rather than non-enzymatic reaction. 

One potential drawback to multi-target screens is that they are unable to distinguish between different modes of inhibition, so are equally likely to identify substrate binders and enzyme binders. The latter are specifically needed to help unravel enzymatic mechanism. Affinity screens can be used to identify compounds that bind to the enzyme. This type of screen has been successfully used to identify inhibitors of MurF that were subsequently used to obtain a crystal structure to aid in drug design [180,181]. A slightly different approach is a displacement screen, which selects for inhibitors that compete with the substrate for binding. This method has been used successfully to identify small-molecule inhibitors of MurG [182,183]. 

The time course of an enzymatic reaction permits one to deduce the substrate affinity, the catalytic mechanism in the active center, and the efficiency of the enzyme (maximum rate, turnover number). 

The reciprocal form of the equation produced a straight line with intercept values on the Y axis of 1/Vmax and on the X axis of -1/Km (Figure 2). This advancement in analysis of the Michaelis-Menten equation allowed for a simplified way of analyzing the effect of compounds that altered the catalytic activity of enzyme systems. Changes in enzymatic activity were observed to result from changes in the substrate affinity or maximum velocity (Lineweaver Burk 1934) resulting in the definition of inhibitory equations based on their effects on the kinetic constants of the Michaelis-Menten equation. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

FIGURE 15.12 71 versus [S] curves for an allosteric V system. The V system fits the model of Moiiod, Wyman, and Chaiigeux, given the following conditions (1) R and T have the affinity for the substrate, S. (2) The effectors A and I have different affinities for R and T and thus can shift the relative T/R distribution. (That is, A and I change the apparent value of L.) Assume as before that A binds only to the R state and I binds only to the T state. (3) R and T differ in their catalytic ability. Assume that R is the enzymatically active form, whereas T is inactive. Because A perturbs the T/R equilibrium in favor of more R, A increases the apparent Vmax- I favors transition to the inactive T state. 

We have just discussed several common strategies that enzymes can use to stabilize the transition state of chemical reactions. These strategies are most often used in concert with one another to lead to optimal stabilization of the binary enzyme-transition state complex. What is most critical to our discussion is the fact that the structures of enzyme active sites have evolved to best stabilize the reaction transition state over other structural forms of the reactant and product molecules. That is, the active-site structure (in terms of shape and electronics) is most complementary to the structure of the substrate in its transition state, as opposed to its ground state structure. One would thus expect that enzyme active sites would bind substrate transition state species with much greater affinity than the ground state substrate molecule. This expectation is consistent with transition state theory as applied to enzymatic catalysis. 

Miller and Wolfenden, 2002). This latter ratio is the inverse of the rate enhancement achieved by the enzyme. In other words, the enzyme active site will have greater affinity for the transition state structure than for the ground state substrate structure, by an amount equivalent to the fold rate enhancement of the enzyme (rearranging, we can calculate KJX = Ksik Jk, )). Table 2.2 provides some examples of enzymatic rate enhancements and the calculated values of the dissociation constant for the /A binary complex (Wolfenden, 1999). 

In Chapter 3 we saw that inhibitors of different modalities respond differently to the concentration of substrate used in an enzymatic reaction. Recall that the apparent affinity of the free enzyme for substrate was diminished in the presence of a competitive inhibitor, and vice versa, the apparent affinity of a competitive inhibitor could be abrogated at high substrate concentrations. On the other hand, the appar-... 

Torres, E. Baeza, A., and Vazquez-Duhalt, R., Chemical modification of heme group improves hemoglobin affinity for hydrophobic substrates in organic media. Journal of Molecular Catalysis, B-Enzymatic, 2002. 19 pp. 437—441. 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) is another recently introduced technique for quantification of proteins, and to date has most often been applied to clinical enzymology.60 The product conjugates of the enzymatic reaction between the synthetic substrate and targeted enzyme are captured by immobilized affinity reagents, purified, released into solution, and analyzed by ESI-MS. 

Turning to enzymatic hydration, we see from the data in Table 10.1 that phenanthrene 9,10-oxide Fig. 10.10, 10.29) is an excellent substrate for epoxide hydrolase. Comparison of enzymatic hydration of the three isomeric phenanthrene oxides shows that the Vmax with the 9,10-oxide is greater than with the 1,2- or the 3,4-oxide the affinity was higher as well, as assessed by the tenfold lower Km value [90]. Furthermore, phenanthrene 9,10-oxide has a plane of symmetry and is, thus, an achiral molecule, but hydration gives rise to a chiral metabolite with high product enantioselectivity. Indeed, nucleophilic attack by epoxide hydrolase occurs at C(9) with inversion of configuration i.e., from below the oxirane ring as shown in Fig. 10.10) to yield the C-H9.S, 10.S )-9,10-dihydro-9,10-diol (10.30) [91],... 

The reduction in enzymatic activity that results from the formation of nonproductive enzyme complexes at high substrate concentration. The most straightforward explanation for substrate inhibition is that a second set of lower affinity binding sites exists for a substrate, and occupancy of these sites ties up the enzyme in nonproductive or catalytically inefficient forms. Other explanations include (a) the removal of an essential active site metal ion or other cofactor from the enzyme by high concentrations of substrate, (b) an excess of unchelated substrate (such as ATP" , relative to the metal ion-substrate complex (such as CaATP or MgATP ) which is the true substrate and (c) the binding of a second molecule of substrate at a subsite of the normally occupied substrate binding pocket, such that neither substrate molecule can attain the catalytically active conformation". For multisubstrate enzymes, nonproductive dead-end complexes can also result in substrate inhibition in the presence of one of the reaction... 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Two different enzymatically active forms of PFK could be identified which may be considered the R and T form in the framework of the symmetry model. The R form possesses a high affinity for the substrate fructose-6-P, the T form binds fructose-6-P with lower affinity. Upon binding of the inhibitor phosphoenolpyruvate, PFK converts to the T form. The enzyme is foimd in the R form upon binding the substrates (ATP or fructose-6-P) or the activator (ADP). There exist high resolution crystal structures of both forms. 

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