Enzymes detailed mechanisms

In Chapter 16, we explore in greater detail the factors that contribute to the remarkable catalytic power of enzymes and examine specific examples of enzyme reaction mechanisms. Here we focus on another essential feature of enzymes the regulation of their aetimty. 

Substrate analogs which promise to be particularly good active-site probes are those which are conformationally restricted. One key feature of enzymatic processes is that when a substrate is bound to an enzyme, probably only one of the many possible conformations of the substrate molecule is assumed. Consequently, before a detailed mechanism for an enzymatic process can be formulated, the preferred conformations of each of the enzyme-bound substrates must be known. ... 

Let us consider an enzymatic reaction in which two substrates are utilized to from two products (in the nomenclature of enzyme reaction mechanisms this situation is referred to as a bi-bi mechanism). A reaction in which one substrate yields two products is referred to as a uni-bi mechanism, and one in which two substrates combine to form a single product is referred to as a bi-uni mechanism (see Copeland, 2000, for further details). For the purposes of illustration let us use the example of a group transfer reaction, in which a chemical species, X, is transferred from one substrate to the other in forming the products of the reaction ... 

Both Vioxx and Celebrex derive their COX2 selectivity from the same type of isozyme-specific slow enzyme isomerization mechanism that was detailed here for DuP697. 

The detailed mechanism of inhibition of TEM-2 (class A) enzyme with clavulanate has been established (Scheme 1) [23,24], The inhibition is a consequence of the instability of the acyl enzyme formed between the /1-lactam of clavulanate and the active site Ser-70 of the enzyme. In competition with deacylation, the clavulanate acyl-enzyme complex A undergoes an intramolecular fragmentation. This fragmentation initially provides the new acyl enzyme species B, which is at once capable of further reaction, including tautomeriza-tion to an entity C that is much less chemically reactive to deacylation. This species C then undergoes decarboxylation to give another key intermediate enamine D, which is in equilibrium with imine E. The imine E either forms stable cross-linked vinyl ether F, by interacting with Ser-130 or is converted to the hydrated aldehyde G to complete the inactivation. 

The detailed mechanism of this reaction has become the subject of vigorous debate since these isotope effects were first published. It has been chosen for detailed theoretical analysis in several QM/MM studies of enzyme catalyzed reactions. 

Knowledge of the detailed mechanism underlying circadian rhythms continues to be refined as new experiments reveal novel facets of the oscillatory machinery. Thus, a link has recently been established between chromatin structure and the circadian oscillatory mechanism. The CLOCK protein indeed functions as a histone acetyltransferase [116]. This enzyme activity is required for oscillations so that histone modification and the associated chromatin remodeling are implicated in the origin of circadian rhythmicity. 

The detailed mechanism of P aeruginosa CCP has been studied by a combination of stopped-flow spectroscopy (64, 65, 84, 85) and paramagnetic spectroscopies (51, 74). These data have been combined by Foote and colleagues (62) to yield a quantitative scheme that describes the activation process and reaction cycle. A version of this scheme, which involves four spectroscopically distinct intermediates, is shown in Fig. 10. In this scheme the resting oxidized enzyme (structure in Section III,B) reacts with 1 equiv of an electron donor (Cu(I) azurin) to yield the active mixed-valence (half-reduced) state. The active MV form reacts productively with substrate, hydrogen peroxide, to yield compound I. Compound I reacts sequentially with two further equivalents of Cu(I) azurin to complete the reduction of peroxide (compound II) before returning the enzyme to the MV state. A further state, compound 0, that has not been shown experimentally but would precede compound I formation is proposed in order to facilitate comparison with other peroxidases. 

Unconsumed substrates are treated as substrates or essential activators in deriving rate equations and studying detailed mechanisms. Nonetheless, one must indicate whether an unconsumed substrate (U) remains bound to the enzyme or not (in this case, U also becomes an unaltered product) in the reaction scheme. In practice, unconsumed substrates are likely to be involved in all the typical multisubstrate kinetic mechanisms Only one case is illustrated here, namely that the unconsumed substrate Su activates catalysis when bound in a rapid-equilibrium ordered mechanism ... 

The overall rate equation of complex single-route reaction with the linear detailed mechanism was derived and analyzed in detail by many researchers. King and Altman (1956) derived the overall reaction rate equation for single-route enzyme reaction with an arbitrary number of intermediates... 

The chemistry of a fourth coenzyme was at least partially elucidated in the period under discussion. F. Lynen and coworkers treated P-methylcrotonyl coenzyme A (CoA) carboxylase with bicarbonate labelled with 14C, and discovered that one atom of radiocarbon was incorporated per molecule of enzyme. They postulated that an intermediate was formed between the enzyme and C02, in which the biotin of the enzyme had become car-boxylated. The carboxylated enzyme could transfer its radiolabelled carbon dioxide to methylcrotonyl CoA more interestingly, they found that the enzyme-COz compound would also transfer radiolabelled carbon dioxide to free biotin. The resulting compound, carboxybiotin [4], was quite unstable, but could be stabilized by treatment with diazomethane to yield the methyl ester of N-carboxymethylbiotin (7) (Lynen et al., 1959). The identification of this radiolabelled compound demonstrated that the unstable material is N-carboxybiotin itself, which readily decarboxylates esterification prevents this reaction, and allows the isolation and identification of the product. Lynen et al. then postulated that the structure of the enzyme-C02 compound was essentially the same as that of the product they had isolated from the reaction with free biotin, but where the carbon dioxide was inserted into the bound biotin of the enzyme (Lynen et al., 1961). Although these discoveries still leave significant questions to be answered as to the detailed mechanism of the carboxylation reactions in which biotin participates as coenzyme, they provide a start toward elucidating the way in which the coenzyme functions. 

The detailed mechanisms for transcription-associated structural changes in chromatin, called chromatin remodeling, are now coming to light, including identification of a variety of enzymes directly implicated in the process. These include enzymes that covalently modify the core histones of the nucleosome and others that use the chemical energy of ATP to remodel nucleosomes on the DNA (Table 28-2). 

The final step in riboflavin biosynthesis has been extensively investigated. Incorporation and degradation studies with synthetic (33) using cell-free systems and purified enzymes have shown that two molecules of (33) are utilized to afford one molecule of riboflavin and one molecule of (36). Significantly, the lumazine (33) labelled at the C-6 methyl with deuterium is converted to riboflavin labelled at C-5 and in the C-7 methyl. Based on this and kinetic and spectroscopic data, Plaut has proposed a detailed mechanism for the riboflavin synthetase reaction (B-71MI10402). It is noteworthy that this reaction can also be accomplished non-enzymatically under neutral conditions with the same stereospecificity observed in the enzymic reaction (69CC290). 

The mode of action, which is now well authenticated and understood, involves the irreversible inhibition of the enzyme acetylcholinesterase, which is essential to nervous conduction in insects, by phosphorylation of a hydroxy group at the active site. The detailed mechanisms have been reviewed by O Brien (B-67MI10700, B-76MI10701). 

A carbanionic intermediate has often been suggested for this enzyme.67 68 However, despite measurements of kinetic isotope effects and many other experiments it has been difficult to establish a detailed mechanism.68... 

Describe the subunit structure of the enzyme pyruvate dehydrogenase. Discuss the functioning of each of the coenzymes that are associated with these subunits and write detailed mechanisms for each step in the pyruvate dehydrogenase reaction. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

None of the detailed mechanisms to be discussed considers the macro-molecular association that may be involved in the action of RNase on high molecular weight polyribonucleotides. Preiss reported from light scattering studies that very large RNA-enzyme aggregates may be formed (393). Their significance for the catalytic mechanism is unknown. 

I n chapter 7 we saw that enzymes can increase the rates of reactions by many orders of magnitude. We noted that enzymes are highly specific in the reactions they catalyze and in the particular substrates they accept. In this chapter we explore the mechanisms of several enzyme-catalyzed reactions in greater detail. Our goal is to relate the activity of each of these enzymes to the structure of the active site, where the functional groups of amino acid side chains, the polypeptide backbone, or bound cofactors must interact with the substrates in such a way as to favor the formation of the transition state. We explore enzyme catalytic mechanisms in many subsequent chapters as well but usually in less detail than here. 

In the preceding sections, we discussed five themes that occur frequently in enzyme reaction mechanisms. We now examine several representative enzymes in finer detail. We focus on three enzymes for which crystal structures have been obtained, because the most decisive advances in our understanding of enzyme reaction mechanisms have come by inspecting such structures. 

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