Chemical modification and amino acid sequence of active site in sugar beet alpha-glucosidase.

The modification of amino acid residues in sugar beet alpha-glucosidase with conduritol B epoxide (CBE), an affinity labeling reagent, inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only the decrease of V values and no change in Km value. An 3H-CBE labeled peptide isolated from the digest of the inactivated enzyme with Lys-C protease was sequenced. The -COO- group of Asp was found to be specifically labeled, implicating that it is a catalytic group of the enzyme. The sequence around the essential Asp was determined to be -DGIWIDMNE-, which showed a high homology with those of other alpha-glucosidases.