Natarajan S, Sierks M R
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 21228, USA.
Biochemistry. 1996 Mar 5;35(9):3050-8. doi: 10.1021/bi952458x.
The functional role of a loop region, highly conserved among glucoamylase and other starch hydrolases which also includes the essential Trp120 of Aspergillus awamori, is investigated. Residues 121-125 of A. awamori glucoamylase were singly substituted, and their individual effects on catalytic activity and thermal stability were determined. The Arg122-->Tyr mutation displayed opposing effects for shorter and longer maltooligosaccharide substrates, K(m) decreasing for shorter substrates but increasing for longer substrates. The Pro123-->Gly mutation decreases the thermal stability of glucoamylase by 19 degrees C with little effect on activity. The Gln124-->His substitution decreases k(cat) for all substrates 10-15-fold. Gly121-->Thr and Arg125-->Lys had only minor effects on glucoamylase activity. While Arg122-->Tyr, Gln124-->His, and the previously constructed Trp120-->Phe [Sierks, M. R., Svensson, B., Ford, C., & Reilly, P. J. (1989) Protein Eng. 2, 621-625] glucoamylases have significantly reduced activity toward maltose hydrolysis, all mutations in the Trp120 loop region retain wild-type level activity toward alpha-D-glucosyl fluoride hydrolysis. The Trp120 loop region therefore plays a major role in directing conformational changes controlling the postulated rate-limiting product release step, even though only Trp120 is indicated to interact with acarbose in the crystal structure [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639]. Side chains of residues 116, 120, 122, and 124 oriented in one direction play crucial roles in the enzyme mechanism, while side chains of residues 119, 121, 123, and 125, oriented in the opposite direction, play only minor roles.
对一个在葡糖淀粉酶和其他淀粉水解酶中高度保守的环区的功能作用进行了研究，该环区还包括泡盛曲霉的必需色氨酸Trp120。对泡盛曲霉葡糖淀粉酶的121 - 125位残基进行了单一位点取代，并测定了它们对催化活性和热稳定性的各自影响。Arg122→Tyr突变对较短和较长的麦芽寡糖底物表现出相反的影响，对于较短的底物K(m)降低，而对于较长的底物K(m)增加。Pro123→Gly突变使葡糖淀粉酶的热稳定性降低了19℃，而对活性影响很小。Gln124→His取代使所有底物的k(cat)降低了10 - 15倍。Gly121→Thr和Arg125→Lys对葡糖淀粉酶活性只有轻微影响。虽然Arg122→Tyr、GGGln124→His以及先前构建的Trp120→Phe [Sierks, M. R., Svensson, B., Ford, C., & Reilly, P. J. (1989) Protein Eng. 2, 621 - 625]葡糖淀粉酶对麦芽糖水解的活性显著降低，但Trp120环区的所有突变对α - D - 葡糖基氟水解仍保持野生型水平的活性。因此，Trp120环区在引导构象变化以控制假定的限速产物释放步骤中起主要作用，尽管在晶体结构中仅显示Trp120与阿卡波糖相互作用 [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631 - 15639]。沿一个方向取向的116、120、122和124位残基的侧链在酶促机制中起关键作用，而沿相反方向取向的119、12,1、123和125位残基的侧链仅起次要作用。
