Probing the role of the chloride ion in the mechanism of human pancreatic α-amylase
Probing the role of the chloride ion in the mechanism of human pancreatic α-amylase
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DOI：
10.1021/bi0115636
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发表时间：
2002-01-08
期刊：
影响因子：
2.9
通讯作者：
Withers, SG
中科院分区：
文献类型：
--
作者：
Numao, S;Maurus, R;Withers, SG
人胰腺α-淀粉酶（HPA）是参与淀粉降解的α-淀粉酶家族的成员。这个家族的一些成员，包括HPA，需要氯化物才能获得最大的活性。为了确定氯离子活化的机制，制备了一系列突变体（R195 A、R195 Q、N298 S、R337 A和R337 Q），其中氯离子结合位点中的残基被替换。发现该结合位点的突变严重影响HPA结合氯离子的能力，而R195和R337突变酶未检测到结合。X射线晶体学分析显示，这些突变并没有导致显着的结构变化。然而，这些突变的引入确实改变了酶的动力学性质。残基R 195的突变导致该酶对淀粉的活性降低20-450倍，并将最适pH值转移到更碱性的pH值。有趣的是，用非碱性氨基酸取代R337导致α-淀粉酶不再需要氯化物进行催化，并且具有与野生型HPA相似的pH曲线。相比之下，在残基N298处的突变导致对氯的结合亲和力低得多的酶，但仍需要氯来获得最大活性。我们提出，需要氯化物来增加酸/碱催化剂E233的pK（a），否则由于R337（一种带正电荷的残基）的存在，pK（a）会更低。
Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family involved in the degradation of starch. Some members of this family, including HPA, require chloride for maximal activity. To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced. Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes. X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes. However, the introduction of these mutations did alter the kinetic properties of the enzyme. Mutations to residue R 195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity. We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue.