INTERMEDIATES, COVALENT ENZYME-SUBSTRATE

PING PONG HALF-REACTIONS. Many enzymes operate by double-displacement mechanisms involving covalent enzyme-substrate intermediates as shown in the following scheme ... 

Recently, the formation of a covalent glycosyl-enzyme intermediate was also shown by Bell and Koshland (17) in another reaction. Evidence was presented that the mechanism of the enzyme, phosphoribosyl-adeno-sine triphosphate pyrophosphate phosphoribosyl transferase, proceeds through a covalent phosphoribosyl-enzyme intermediate. The intermediate has been demonstrated after incubating the enzyme with 14C-5-phosphoribosyl-l-pyrophosphate (PRPP) under native and denaturing conditions. The intermediate also forms from the reverse direction as shown when the enzyme is mixed with its product N- (5-phosphoribosyl-adenosine triphosphate (PR-ATP). These data give evidence for a covalent enzyme-substrate intermediate. The enzyme which catalyzes the overall reaction proceeds as follows ... 

It is reasonable to identify the intermediate indicated by the above-mentioned experiments as a y-glutamyl-enzyme compound, an interpretation not excluded by any of the experimental results. There is, however, another plausible explanation for the observations, which does not necessarily involve a covalent enzyme-substrate compound of this kind. In this alternative proposal the rate determining steps in the catalytic reaction are not involved with the covalent bond processes but are conformational changes in the enzyme-substrate and enzyme-product complexes. If product is not released from the enzyme until a large number of rapid covalent reactions with the available nucleophiles has occurred, then any substrate will be converted to the same equilibrium mixture of bound products (e.g., glutamic acid and glutamyl hydroxamic... 

In a review of the proficiencies of enzymes and how they achieve them, it was claimed that ground-state conformations and transition state stabilization cannot explain the very large efficiencies of enzymes instead, they must proceed, it was concluded, by covalent enzyme-substrate intermediates.73 A riposte to this contentious hypothesis has appeared, claiming that account was not taken of the fact that high enzyme efficiency is determined by the value for the water reaction k0 rather than by the enzymatic rate constant kcat/kM.14... 

The mechanism by which influenza virus sialidases cleaves the Neu5Ac(a2 3)Gal or Neu5Ac(a2- 6)Gal linkage has been a topic of much interest for many years (e.g., see [71, 72]). Recently, it has been shown by structural analysis [73] that it involves a covalent enzyme-substrate intermediate as has been reported for other sialidases. A proposed mechanism is depicted in Scheme 17.1. 

Kuroki, R., Weaver, L. H., and Matthews, B. W. A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme. Science 262, 2030-2033 (1993). 

Transfer of a two-carbon unit from a 2-keto sugar to the carbonyl carbon (Ci) of an aldose by a transketolase, which requires thiamine pyrophosphate and magnesium as cofactors. A covalent enzyme-substrate intermediate is formed similar to the one that occurs during the pyruvate dehydrogenase reaction (Chapter 13). 

For all the enzymes investigated to date, inversion of configuration has always been observed for reactions known to be single-step processes. For reactions known to involve two steps (via the formation of a covalent enzyme-substrate intermediate), the stereochemical result is always retention. These results suggest that the general mechanism of enzyme-catalyzed phosphoryl transfer reactions always involves inversion, which rules out the adjacent pathway D but does not differentiate the dissociative pathway and the in-line associative pathway. 

One type of evidence often presented in support of double-displacement mechanisms involving covalent enzyme-substrate intermediates is the observation of exchange reactions of the type discussed above for galactose-1-P uridylyltrans-ferase and acetate kinase. The exchange reactions (19a) and (19b), where PGA is 3-phosphoglycerate and DPG is 1,3-diphosphoglycerate, are catalyzed by phosphoglycerate kinase (68, 70). 

The term commitment to catalysis " is unique to KIE studies on enzymes and not widely used in the chemical literature. External commitment to catalysis refers to the partitioning of the hrst intermediate of the reaction, the non-covalent enzyme-substrate (E-S) intermediate. Some enzymes are such efficient catalysts that nearly every molecule of substrate that binds proceeds forward to products (i.e. fes > 2)- Under these conditions, the hrst irreversible step of the reaction is substrate binding. If competitive isotope effects are measured, they will only reflect binding isotope effects, and have no contribution from the chemical steps of the reaction. In less extreme cases (Eig. 2d), both substrate binding ( 1) and the chemical step ( 3) are partially irreversible. Internal commitment to catalysis simply refers to the partitioning of any intermediate enzyme-bound species. 

The noncovalent or covalent enzyme-substrate, enzyme-intermediate and enzyme-product species are detected based on the miz ratio... 

Strong evidence from mass spectrometry su ests that the mechanism of lysozyme involves sequential 5 2 reactions and a covalent enzyme—substrate intermediate (based on work by Stephen Withers and colleagues at the University of British Columbia and elsewhere). Asp-52 acts as the nucleophile in the first step, covalently bonding the substrate to the enzyme. A water molecule acts as a nucleophile in the second step to complete the formation of product and free the substrate from the active site. In both steps, Glu-35 serves as a general acid—base catalyst. The details are as follows. 

FIGURE 24.17 The double-displacement Sn2 mechanism for lysozyme shown here is supported by mass spectometric evidence for a covalent enzyme-substrate intermediate. 

Many enzymes form covalent bonds with their substrates during catalysis, such as the acyl-enzyme intermediate in carboxyl ester hydrolysis (Scheme 2.1) or the glycol monoester intermediate in epoxide hydrolysis (Scheme 2.85). Despite the covalent enzyme-substrate bond, such species are metastable and should be regarded as activated intermediates . Some enzymes utilize cofactors, such as... 

Rudolph and Fromm 12) have recently made a thorough kinetic study of this enzyme and found the binding of substrates and dissociation of products to be random however, covalent enzyme-substrate intermediates did not take part in the reaction. They concluded that it is still not possible to decide between the mechanism proposed by Lieberman (11), involving a 6-phosphoryl inosinate intermediate, and the simultaneous interaction of the reactants in a concerted transition complex as suggested by Miller and Buchanan (13). 

Paracatalytic enzyme modification is a new type of catalysis-linked and, hence, substrate-dependent enzyme modification. In all instances in which the substrate promotes inactivation of an enzyme by a chemical reagent, particularly by an oxidant, paracatalsrtic modification should he considered to be the underlying mechanism. In contrast to ligand-induced and syncatalytic modifications, paracatalytic modification involves a direct chemical interaction between enzyme-activated substrate and extrinsic reagent. In this respect, it is similar to the chemical trapping of covalent enzyme-substrate intermediates, e.g., the reduction of enzyme-substrate Schiff bases by sodium borohydride in class I fructose-l,6-bis-phosphate aldolases - or in acetoacetate decarboxylase. ... 

A number of other enzymes which catalyze the hydrolysis of phosphoesters are of biological importance. These include cyclic purine phosphodiesterase (little is known about its active site chemistry at present, but more shall be said about its biological role shortly) and the phosphatases. Acid and alkaline phosphatase catalyze the hydrolysis of phosphomonoesters to the corresponding alcohol and inorganic phosphate. Their pH optimums are 5.0 and 8.0, respectively hence their names. Both form covalent enzyme-substrate intermediates ... 

Alternatively, the enzyme may first be phosphorylated by the ATP to form a transient covalent enzyme-substrate intermediate, which then suffers displacement by the substrate. The net result is a retention of configuration or two inversion processes ... 

Chymotrypsin is the most-studied member of the serine protease family of enzymes. The enzyme-catalysed hydrolytic reaction has been shown to occur in at least three kinetically distinguishable steps. The first of these consists of a very fast, diffusion-controlled formation of a non-covalent enzyme-substrate complex, followed by an acylation step. In the latter the acyl group of the substrate is covalently attached to a serine alcohol of the active site with the concomitant release of the amine of an amide substrate, or the alcohol of an ester substrate. In a final deacylation step the acyl-enzyme intermediate is hydrolysed by water, thus regenerating the free enzyme and releasing the carboxylic acid ... 

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