Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase
Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase
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DOI：
10.1128/jb.187.7.2386-2394.2005
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发表时间：
2005-04-01
影响因子：
3.2
通讯作者：
Ferry, JG
中科院分区：
文献类型：
--
作者：
Ingram-Smith, C;Gorrell, A;Ferry, JG
醋酸激酶通过将三磷酸腺苷的γ-磷酰基转移到醋酸酯上，催化可逆的依赖镁的乙酰磷酸的合成。对只含ADP的嗜热甲烷八叠球菌酶的晶体结构的研究发现，活性部位裂隙中的Val(93)、Len(122)、Phe(179)和Pro(232)残基形成了一个可溶于溶剂的疏水口袋，从而确定了一个潜在的醋酸酯结合部位。这是一个结合位点的假设进一步得到了数据库中所有醋酸激酶序列的比对，这表明所有四个残基都是严格保守的，以及嗜热分枝杆菌酶的最新晶体结构与这个口袋中的醋酸盐结合。替换口袋中的每个残基产生了醋酸酯的K-m值比野生型大7到26倍的变体，这种结合口袋的扰动也改变了较长链羧酸和乙酰磷酸的专一性。变异体的动力学分析结合结构模型表明，该口袋具有结合乙酸甲酯、影响底物专一性和定向羧基的作用。动力学分析还表明，乙酰磷酸的结合更依赖于磷酸基团与未知残基的相互作用，而不是甲基与疏水口袋之间的相互作用。分析还表明，Phe(179)对催化是必不可少的，可能是结构域关闭所必需的。从数据库中获得的醋酸激酶、丙酸激酶和丁酸激酶序列的比对表明，这些酶具有相似的催化机制和羧酸底物结合位点。
Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Len(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K-m values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.