Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, Bphl
Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, Bphl
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DOI：
10.1021/bi101947g
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发表时间：
2011-05-03
期刊：
影响因子：
2.9
通讯作者：
Seah, Stephen Y. K.
中科院分区：
文献类型：
--
作者：
Baker, Perrin;Carere, Jason;Seah, Stephen Y. K.
BphI是多氯联苯（PCBs）降解途径中的一种特异性H类醛缩酶，催化（4S）-羟基-2-含氧酸的可逆C-C键断裂形成丙酮酸和醛。突变是。引入到bphI探针活性位点残基的底物识别和催化的贡献。与对乙醛和丙醛具有相似特异性的野生型酶相反，L87A变体对丙醛的偏好是乙醛的40倍。在使用戊醛的羟醛加成反应中，L89A变体的特异性常数增加了约50倍;使得与天然底物乙醛的野生型利用相比，其对于戊醛利用更催化有效。用苯丙氨酸或丝氨酸替换Tyr-290导致立体化学控制的丧失，因为变体能够利用具有类似动力学参数的C4处的R和S构型的底物。在R16A变体中未检测到羟醛裂解和丙酮酸α-质子交换活性，支持Arg-16在稳定丙酮酸烯醇化物中间体中的作用。该酶的pH依赖性与pK（a）值约为7的催化碱的单一去质子化一致。在H20A和H20S变体中，pH曲线显示酶活性对氢氧化物浓度的依赖性。在此基础上，提出了催化机理。
BphI, a pyruvate-specific class H aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde.. Mutations were. introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the. L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde.,The, specificity Constant of the L89A variant in the aldol addition reaction using pentaldehyde is, increased similar to 50-fold; making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S:configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate alpha-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH :dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A, and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.