Covalently bound intermediates, catalytic reactions

A catalytic cycle is composed of a series of elementary processes involving either ionic or nonionic intermediates. Formation of covalently bound species in the reaction with surface atoms may be a demanding process. In contrast to this, the formation of ionic species on the surface is a facile process. In fact, the isomerization reaction, the hydrogenation reaction, and the H2-D2 equilibration reaction via ionic intermediates such as alkyl cation, alkylallyl anion, and (H2D)+ or (HD2)+ are structure-nonrequirement type reactions, while these reactions via covalently bound intermediates are catalyzed by specific sites that fulfill the prerequisites for the formation of covalently bound species. Accordingly, the reactions via ionic intermediates are controlled by the thermodynamic activity of the protons on the surface and the proton affinity of the reactant molecules. On the other hand, the reactions via covalently bound intermediates are regulated by the structures of active sites. 

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... 

These biochemical transformations occur on a multienzyme complex composed of at least three dissimilar proteins biotin carrier protein (MW = 22,000), biotin carboxylase (MW = 100,000) and biotin transferase (MW = 90,000). Each partial reaction is specifically catalyzed at a separate subsite and the biotin is covalently attached to the carrier protein through an amide linkage to a lysyl a-amino group of the carrier protein (338, 339). In 1971, J. Moss and M. D. Lane, from Johns Hopkins University proposed a model for acetyl-CoA carboxylase of E, coli where the essential role of biotin in catalysis is to transfer the fixed CO2, or carboxyl, back and forth between two subsites. Consequently, reactions catalyzed by a biotin-dependent carboxylase proceed though a carboxylated enzyme complex intermediate in which the covalently bound biotinyl prosthetic poup acts as a mobile carboxyl carrier between remote catalytic sites (Fig. 7.13). 

Most EHs have a/ 3-hydrolase fold topology and consist of a core and a lid domain [65,66]. The lid domain is mainly a-helical and contains two tyrosine residues that point toward the catalytic triad and cover the core domain. Both tyrosine residues are involved in substrate binding, Uansition-state stabilization, and activation of the epoxide by protonation. The catalytic center is composed of two aspartate and one histidine residue. The first crystal structure of an epoxide hydrolase was solved for the enzyme from Agrobacterium radiobacter ADI (EchA) [67]. The reaction mechanism of EHs is depiaed in Scheme 9.9. First, a nucleophilic attack of the aspartic residue on the epoxide ring of the substrate 31 takes place and a covalently bound ester 32 is formed. This intermediate is subsequently hydrolyzed by a so-called charge relay system (general base catalysis) and the diol 33 is released from the active site. Key reaction parmers are a histidine residue and a water molecule. It is worth mentioning that a limonene epoxide hydrolase discovered by Arand et al. displayed a different crystal structure and catalytic cycle that is discussed elsewhere [68]. 

The Michaelis-Menten scheme may be extended to cover a variety of cases in which additional intermediates, covalently or noncovalently bound, occur on the reaction pathway. It is found in all examples that the Michaelis-Menten equation still applies, although KM and cataTemqwcombinations of various rate and equilibrium constants. KM is always less than or equal to Ks in these cases. Suppose that, as for example in the following scheme, there are several intermediates and the final catalytic step is slow ... 

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