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首页> 外文期刊>Biochemistry >FUNCTIONAL AND STRUCTURAL ROLES OF THE HIGHLY CONSERVED TRP120 LOOP REGION OF GLUCOAMYLASE FROM ASPERGILLUS AWAMORI
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FUNCTIONAL AND STRUCTURAL ROLES OF THE HIGHLY CONSERVED TRP120 LOOP REGION OF GLUCOAMYLASE FROM ASPERGILLUS AWAMORI

机译：泡盛曲霉葡糖淀粉酶的高度保守的TRP120循环区域的功能和结构作用

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摘要

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., Br 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.
机译：研究了在葡糖淀粉酶和其他淀粉水解酶之间高度保守的环状区域的功能性作用，其中还包括泡盛曲霉（Aspergillus awamori）的必需Trp120。泡盛曲霉葡糖淀粉酶的残基121-125被单独取代，并确定了它们对催化活性和热稳定性的单独影响。 Arg122-> Tyr突变对于较短和较长的麦芽低聚糖底物表现出相反的作用，对于较短的底物，K-m减小，而对于较长的底物，K-m减小。 Pro123-> Gly突变使葡糖淀粉酶的热稳定性降低了19摄氏度，而对活性的影响很小。对于所有底物，Gln124-> His取代都会使k（cat）降低10-15倍。 Gly121-> Thr和Arg125-> Lys对葡糖淀粉酶活性的影响很小。而Arg122-> Tyr，Gln124-> His和先前构建的Trp120-> Phe [Sierks，M. R.，Svensson，B.，Ford，C.，＆Reilly，P.J.（1989）Protein Eng。 2，2，621-625]葡糖淀粉酶对麦芽糖水解的活性显着降低，Trp120环区域中的所有突变都保留了对α-D-D-葡糖基氟化物水解的野生型水平活性。因此，即使表明仅Trp120与晶体结构中的阿卡波糖相互作用，Trp120环区域在指导构象变化控制假定的限速产物释放步骤中也起着重要作用[Aleshin，AE，Firsov，LM，Br Honzatko，RB （1994）J.Biol。化学269，15631-15639]。沿一个方向取向的残基116、120、122和124的侧链在酶机理中起关键作用，而沿相反方向取向的残基119、121、123和125的侧链仅起较小作用。

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