CATALYTIC MECHANISM OF GLUCOAMYLASE PROBED BY MUTAGENESIS IN CONJUNCTION WITH HYDROLYSIS OF ALPHA-D-GLUCOPYRANOSYL FLUORIDE AND MALTOOLIGOSACCHARIDES

The catalytic mechanism of glucoamylase (GA) is investigated by compar ing kinetic results obtained using alpha-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116-->Ala, Trp120-->Phe, Asp176-->Asn, and Glu400-->Gl n. These replacements decreased the activity (k(cat)/K-M) toward malto se by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GA s, showed wild-type and twice wild-type level activity, while Asp176-- >Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respect ively. Glu400, the catalytic base, is suggested to strengthen ground-s tate binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr 116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabiliz ation. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp12 0 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limitin g aglycon product release was suggested previously for GA-catalyzed hy drolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & He hre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild-type GA complexed with GF, the pH-activity (k(cat)) profile show s a pK(a) of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu 400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M . R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13 816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the depro tonation of a carboxyl group that facilitates catalysis.