Two Subsites on the Active Center of Pig Kidney Trehalase

A kinetic analysis of the active site of pig kidney trehalase was made by examining two types of inhibitors that are monosaccharide analogs and cause a Competitive inhibition of the trehalase. Trehalase hydrolyzes trehalose (α‐ d ‐glycopyranosyl α‐ d ‐glucopyranoside) to give an equimolar mixture of α‐ d ‐glucose and, by inversion of configuration, β‐ d ‐glucose. 1,4‐Dideoxy‐1,4‐imino‐ d ‐arabinitol is considered to be a transition state (glucosyl cation) analog, while methyl β‐ d ‐glucoside, 1,5‐dideoxy‐1,5‐imino‐ d ‐glucitol (1‐deoxynojirimycin), fagomine, and 1‐epivalidamine are considered to be analogs of the β‐ d ‐glucose that is derived by hydrolysis of trehalose. These glucosyl cation inhibitor and β‐ d ‐glucose analog inhibitors competed with each other at the same site on the active center of pig kidney trehalase and were therefore put together in one group (group A). Methyl α‐ d ‐mannoside and 1‐deoxymannojirimycin were also competitive inhibitors of trehalase and competed with each other for the same site. However, an inhibitor in group A did not compete with the methyl α‐ d ‐mannoside or 1,5–dideoxy‐1,5‐imino‐ d ‐mannitol (1‐deoxymannojirimycin). Thus these latter two inhibitors were placed in group B. These results support the hypothesis that the active center of trehalase may comprise two subsites, one for catalysis and one for recognition, that act separately on each of the glucoses of the trehalose. The catalysis site requires the correct d ‐glucose configuration at carbons 2, 3, 4, and 5 or a good superimposition onto the glucosyl cation intermediate. The C2 equatorial OH group of a glucopyranosyl residue appears to be important for binding at the catalytic site since 1‐deoxynojirimycin is more tightly bound by two orders of magnitude over its 2‐deoxy derivative, fagomine. The β‐ d ‐glucose and glucosyl cation analogs best fit this site. The recognition site is compatible with d ‐glucose and its analogs bearing the α configuration at the anomeric position. α‐ d ‐Mannose analogs are much more tightly bound than the corresponding d ‐ gluco compound at this site. The extremely high affinity ( K i = 0.52 nM) of validoxylamine A, a mimic of the substrate in the transition state, derives from the synergistic interactions of two cyclitol units with two subsites. The value obtained by multiplying the K i (1.2 μM) for 1‐epivalidamine times that for 1‐deoxymannojirimycin ( K i = 0.39 mM) is very close to that for validoxylamine A. The results described here may be applicable to other trehalase molecules.