No abstract

No abstract

The following kinetic isotope effects (klighf/khcavy) have been measured by the isotopic quasi-racemate method for the hydrolyses of methyl a-and 8-glucopyranosides, respectively in 2.0 M HCIOl at 80 "C (a-D 1.137 f 0.007, 1.089 f 0.006; 13-D 1.073 f 0.003, 1.045 f 0.004; y-D (C5) 0.987 f 0.002, 0.971 f 0.003; leaving group d, 1.006 f 0.001, 1.015 f 0.002; leaving group '*O 1.026 f 0.001, 1.024 f 0.001; ring I8O 0.9965 f 0.001, 0.991 f 0.002; anomeric I3C 1.007 f 0.001, 1.01 1 f 0.002).in conjunction with studies of the effect of added solutes on the rates of hydrolysis of various aldopyranosyl derivatives, which indicate such reactions are truely unimolecular, and model ring and @deuterium effects, it is possible to locate the dihedral angles about the 0 5 4 1 and CI-C2 bonds at the transition state using these data. If the dihedral angles so derived are used as constraints in calculations using N.L. Allinger's MM2 molecular mechanics program, transition-state structures are obtained. The geometry of these transition states stands in contradiction to the "theory of stereoelectronic control".Glycosyl transfer is a biologically important process.' This being so, the physical organic chemistry of acetal and of glycoside hydrolysis has received much Although the basic features of the mechanism of acid-catalyzed hydrolysis of glycopyranosides were established over 20 years ago,4 there remain unanswered questions with regard to the timing of bond-making and bond-breaking processes and with regard to stereochemistry, which are particularly pertinent to considerations of enzymic glycosyl t r a n~f e r .~ Since isotopic substitution does not in general alter the potential energy surface for a reaction,6 kinetic isotope effects are the ideal method for addressing these questions. We accordingly elected to measure the kinetic isotope effects on the acid-catalyzed hydrolyses of methyl CY-and P-glucopyranosides, corresponding to the isotopic substitution pattern as shown in structure I. IThe acid-catalyzed hydrolysis of glucopyranosides is known4 to proceed by specific acid catalysis, the exocyclic oxygen atom being reversibly protonated and then the Cl-oxygen bond cleaving. Since the leaving group oxygen atom is completely protonated before glycone-aglycone fission begins, the magnitude of the leaving group lSO kinetic isotope effect is a direct measure of the extent to which the glycone-aglycone bond is broken at the transition state.' Any ring lSO effect will arise because the endocyclic C1-0 bond acquires double-bond character at the transition state as the positive charge on C1 is delocalized by one of the oxygen lone pairs of electrons. It will thus be inverse ( k l 6 / k I s < I.()), and its magnitude will reflect the degree of double-bond character of the C1-0 bond and hence the charge on oxygen. The magnitude of the I3C kinetic isotope effect will, in the absence of nulceophilic participation, reflect the degree to which breaking of the glycone-aglycone bond is compensated for by the tightening C1-05 bond.6There are...

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

No abstract