Renal trehalase: two subsites at the substrate-binding site.
Phlorizin, phloretin, Tris and beta-methylglucoside are competitive inhibitors, with respect to the substrate trehalose, of purified renal trehalase. Mercuric chloride is a noncompetitive inhibitor. The active site of trehalase was examined further by multi-inhibition kinetic studies involving combinations of inhibitors. Phlorizin vs. phloretin and phlorizin vs. Tris were mutually non-competitive. In contrast, phloretin vs. Tris was mutually competitive. These findings suggest that the binding site of phlorizin to the enzyme differed from that of phloretin or Tris, and that phloretin and Tris might bind at a common site. These findings suggest a model in which trehalase has two binding sites at the substrate-binding site, a phlorizin (glucosyl) and a phloretin (phenyl) binding site, analogous to the model proposed previously for the glucose carrier. In addition, mercuric chloride vs. beta-methylglucoside was mutually competitive, although mercuric chloride and beta-methylglucoside, respectively, were noncompetitive and competitive inhibitors with respect to the substrate. Thus, it is suggested that the substrate binding and the SH-inhibitor binding sites are located very close to each other.