Glycosyl-enzyme intermediate

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. 

Group (c), a-D-mannosidase from jack beans and from almonds, and a-D-galactosidase from coffee beans, showed no inactivation. The results with these enzymes can possibly be explained by the formation of a (weak) non-covalent complex in which glycosylation is too slow to cause inactivation within the time period of measurements, or, less likely, rapid hydrolysis of the glycosyl-enzyme intermediate. 

Evidence for a glycosyl-enzyme intermediate of finite lifetime with inverting a-D-glycosidases, and details of its reaction, came from studies with 2,6-anhydro-l-deoxyhept-l-enitols and glycosyl fluorides. - Analysis of hydration and hydrolysis products on the one hand, and of glycosyla-tion products on the other, indicated an intermediate that could be approached by water from the yff-face only of the ring, and by other glycosyl acceptors only from the a-face (see Schemes 4 and 5 This can be considered a proof of the principle of microscopic reversibility of chemical reactions. 

BURMEISTER, W.P., COTLAZ, S, DRIGUEZ, H, LORI, R., PALMIERI, S., HENRISSAT, B., The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an. S -glycosidase, Structure, 1997, 5, 663-675. 

If aspartic acid-52 acts as a nucleophile in lysozyme reactions a glycosyl enzyme intermediate will be formed [60]. There is no evidence, kinetic or otherwise, for substituted enzyme intermediates, but rapid breakdown might preclude attainment of detectable concentrations. Formation of a substituted enzyme could explain the observed retention of configuration at the anomeric carbon in transglycosidation reactions, provided backside attack in a subsequent reaction is chemically reasonable. It has therefore been important to attempt to understand the chemistry of acylal hydrolysis so as to assess the properties that would be expected of an acylal intermediate in reactions catalysed by the enzyme. 

Identification of glycosyl-enzyme intermediates. Studies with pure enzymes often make it possible to confirm directly the existence of enzyme-bound intermediates. The intermediates detected are frequently glycosyl esters of glutamate or aspartate side chain... 

Glycosyl enzyme intermediates 595 Glycosyl esters 595 Glycosyl exchange reactions 593 Glycosylphosphatidylinositol (GPI) anchors 403... 

This model makes no assumption as to detailed reaction mechanism, but it does allow for glycosyl-proton interchange with enzyme (as H-X ) to form glycosyl-enzyme intermediates, whether covalently linked or of carbonium-oxonium type (42). In the past few years such glycosyl-enzyme intermediates have been demonstrated for a number of carbo-... 

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 ... 

Two glycosyl-enzyme intermediates have thus far been definitely... 

If the nature and stereochemistry of the glycosyl-enzyme intermediates have been assigned correctly, therefore, extraordinary conformational subtlety is required of retaining glycosidases. This is difficult to reconcile in evolutionary terms with retention being the commonest stereochemical outcome of glycosidase action. 

In oligosaccharide synthesis via transglycosylation the reaction is kinetically controlled, and the extent of oligosaccharide formation depends on the partition ratio of the glycosyl-enzyme intermediate between the transfer and hydrolytic reaction (Scheme 1). 

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