Sierks M R, Svensson B
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21228, USA.
Biochemistry. 1996 Feb 13;35(6):1865-71. doi: 10.1021/bi951738+.
The catalytic mechanism of glucoamylase (GA) is investigated by comparing 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-->Gln. These replacements decreased the activity (kcat/KM) toward maltose by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GAs, showed wild-type and twice wild-type level activity, while Asp176-->Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 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 stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild-type GA complexed with GF, the pH-activity (kcat) profile shows a pKa of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis.
通过比较以α-D-葡萄糖基氟化物（GF）和麦芽低聚糖为底物时野生型及4种活性位点突变型葡糖淀粉酶（GA）（Tyr116→Ala、Trp120→Phe、Asp176→Asn和Glu400→Gln）的动力学结果，对葡糖淀粉酶（GA）的催化机制进行了研究。这些取代使对麦芽糖的活性（kcat/KM）降低了6至320倍。然而，对于GF，Tyr116→Ala和Trp120→Phe突变型GA表现出野生型及两倍野生型水平的活性，而Asp176→Asn和Glu400→Gln突变型GA的活性分别降低了22倍和665倍。催化碱Glu400被认为可增强亚位点1中的基态结合，而Asp176在亚位点1和2中起此作用。Tyr116和Trp120属于一个芳香簇，该芳香簇与催化位点稍有距离，对GF水解并不关键，但可能参与麦芽低聚糖过渡态的稳定。由于催化位点附近基团的突变降低了对GF和麦芽糖的活性，但Tyr116和Trp120的取代仅降低了对麦芽糖的活性，因此与底物糖苷配基部分的相互作用可能与限速步骤有关。先前已提出GA催化水解的限速步骤是糖苷配基产物的释放[北畑，S.，布鲁尔，C.F.，根霍夫，D.S.，泽井，T.，& 赫雷，E.H.（1981年）《生物化学杂志》256，6017 - 6026]。对于与GF复合的Glu400→Gln和野生型GA，pH - 活性（kcat）曲线显示pKa为2.8。然而，当这两种酶与麦芽糖复合时，只有野生型GA有一个可滴定的碱基基团，确定为Glu400[弗兰森，T.P.，杜邦，C.，莱姆贝克，J.，斯托弗，B.，西克斯，M.R.，洪扎特科，R.B.，& 斯文森，B.（1994年）《生物化学》33，13808 - 13816]。因此，GF与Glu400→Gln GA的结合大概引发了一个促进催化的羧基的去质子化。
