Direct catalysis glycosylation

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

Cytidine 5 -phospho-A-acetylneuraminate (298), the coenzyme of sialyltransferases, is a sugar-nucleotide in which the leaving group is a nucleotidyl monophosphate that contains a carboxylate group directly attached to the anomeric centre. Studies of its hydrolysis reveal that at pH 5 specific acid catalysis occurs (Scheme 34) to yield a glycosyl carbocation as a tight ion pair, the lifetime of which was estimated... 

The alternative role for direct involvement of aspartic acid-52 is electrostatic stabilization of an oxocarbonium ion, a reasonable expectation in view of the steric situation in the active site, the poor internal stabilization of a glycosyl carbonium ion, and the apparent need for some kind of stabilization if general acid catalysis by glutamic acid-35 is to occur. Nevertheless, this assignment is questionable since electrostatic stabilization has not been demonstrable with o-methoxymethoxyisophthalic acid (Dunn and Bruice, 1970) and benzaldehyde disalicyl acetals (Anderson and Fife, 1973). In the former example, such stabilization should be favourable because the carbonium ion intermediate is unstable. 

There is no direct evidence on the form of the glycosyl-enzyme intermediate. Nonetheless, stabilization of a noncovalent oxocarbonium for the period between product fructose release and acceptor binding may not be realistic for slow reactions, given the extremely short lifetime of a glycosyl oxocarbonium ion (9). Levansucrase and particularly GTase-S are quite slow enzymes, with sucrose hydrolysis Heat of 48 sec 122) and 9.1 see" 213), respectively. Thus, the carbonium ion may well collapse to a more stable covalent complex or develop an equilibrium between the two forms. Nucleophilic catalysis is consistent with... 

Enzyme intermediates trapped by chemical modification can provide pertinent details about the enzyme active site and catalytically significant amino acids that directly reflect on the reaction mechanism. However, when the chemical modification is irreversible, demonstrating kinetic relevance by intermediate transfer along the remainder of the reaction pathway at a rate consistent with catalysis is not possible. Thus, distinguishing an authentic covalent intermediate from a collapsed form of a glycosyl-cation is not possible. 

Nevertheless, in the reactions of formation of glycosyl bonds takes always place the acid catalysis, which is connected with effects of protoning of heteroatoms, directly coimected with the glycoside center, for example ... 

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