2 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 hydroly zes trehalose (alpha-D-glycopyranosyl alpha-D-glucopyranoside) to give an equimolar mixture of alpha-D-glucose and, by inversion of configur ation, beta-D-glucose. 1,4-Dideoxy-1,4-imino-D-arabinitol is considere d to be a transition state (glucosyl cation) analog, while methyl beta -D-glucoside, 1,5-dideoxy-1,5-imino-D-glucitol (1-deoxynojirimycin), f agomine, and 1-epivalidamine are considered to be analogs of the beta- D-glucose that is derived by hydrolysis of trehalose. These glucosyl c ation inhibitor and beta-D-glucose analog inhibitors competed with eac h other at the same site on the active center of pig kidney trehalase and were therefore put together in one group (group A). Methyl alpha-D -mannoside and 1-deoxymannojirimycin were also competitive inhibitors of trehalase and competed with each other for the some site. However, an inhibitor in group A did not compete with the methyl alpha-D-mannos ide or 1,5-dideoxy-1,5-imino-D-mannitol (1-deoxymannojirimycin). Thus these latter two inhibitors were placed in group B. These results supp ort the hypothesis that the active center of trehalase may comprise tw o subsites, one for catalysis and one for recognition, that act separa tely on each of the glucoses of the trehalose. The catalysis site requ ires the correct D-glucose configuration at carbons 2, 3, 4, and 5 or a good superimposition onto the glucosyl cation intermediate. The C2 e quatorial OH group of a glucopyranosyl residue appears to be important for binding at the catalytic site since 1-deoxynojirimycin is more ti ghtly bound by two orders of magnitude over its 2-deoxy derivative, fa gomine. The beta-D-glucose and glucosyl cation analogs best fit this s ite. The recognition site is compatible with D-glucose and its analogs bearing the a configuration at the anomeric position. alpha-D-Mannose analogs are much more tightly bound than the corresponding D-gluco co mpound at this site. The extremely high affinity (K-i = 0.52 nM) of va lidoxylamine A, a mimic of thr substrate in the transition state, deri ves from the synergistic interactions of two cyclitol units with two s ubsites. The value obtained by multiplying the K-i (1.2 mu M) for 1-ep ivalidamine times that for 1-deoxymannojirimycin (K-i = 0.39 mM) is ve ry close Co that for validoxylamine A. The results described here may be applicable to other trehalase molecules.