ON THE WATER-PROMOTED MECHANISM OF PEPTIDE CLEAVAGE BY CARBOXYPEPTIDASE-A - A THEORETICAL-STUDY
ON THE WATER-PROMOTED MECHANISM OF PEPTIDE CLEAVAGE BY CARBOXYPEPTIDASE-A - A THEORETICAL-STUDY
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DOI：
10.1139/v94-264
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发表时间：
1994-10-01
期刊：
影响因子：
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通讯作者：
AVILES, FX
中科院分区：
文献类型：
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作者：
ALVAREZSANTOS, S;GONZALEZLAFONT, A;AVILES, FX
用半经验量子力学方法研究了水促进的羧肽酶A裂解多肽的途径。一个相对较大的模型CPA-活性位点元素加底物已被设计，使用两个咪唑和一个乙酸作为Zn 2+配体，乙酸作为质子受体（模拟Glu-270），和N-乙基乙酰胺作为肽样底物。这个模型，虽然比自然的简单，是一个最大的用于CPA催化机制的理论计算。为了确保该模型能够模拟天然系统，将其与由几种分子动力学/能量最小化模拟得到的（Gly）（3）-L-Tyr +水+ CPA复合物的结构进行了比较。在Lipscomb小组提出的水促进途径中涉及的不同步骤中，来自活化水分子的氧原子对底物的肽键的碳原子的攻击被发现是速率决定步骤，具有37.9 kcal/mol的高焓势垒。然而，当模拟Arg-127的正电荷被包括在易分裂的羰基附近时，该焓势垒急剧降低。报道的结果似乎有利于发生的机制研究，并表明使用简单的元素的酶催化反应的理论分析的局限性。
The water-promoted pathway of peptide cleavage by carboxypeptidase A has been studied by semiempirical (AMI) quantum mechanical calculations. A relatively large model for the CPA-active site elements plus substrate has been designed, using two imidazoles and one acetate as the Zn2+ ligands, acetate as the proton acceptor (simulating Glu-270), and N-ethylacetamide as the peptide-like substrate. This model, although simpler than the natural one, is one of the largest used for theoretical calculations on CPA catalytic mechanisms. To ensure that this model is able to mimic the natural system, it has been compared with the structure of the (Gly)(3)-L-Tyr + water + CPA complex resulting from several molecular dynamics/energy minimization simulations. Among the different steps involved in the water-promoted pathway proposed by Lipscomb's group, the attack of the oxygen atom that comes from the activated water molecule to the carbon atom of the peptide bond of the substrate has been found to be the rate-determining step, with a high enthalpy barrier of 37.9 kcal/mol. However, this enthalpy barrier is dramatically decreased when a positive charge, simulating Arg-127, is included near the scissile carbonyl. The reported results seem to favour the occurrence of the mechanism studied and indicate the limitations of using simple elements for the theoretical analysis of enzyme-catalyzed reactions.