Inhibition of chymotrypsin by a complex of ortho-vanadate and benzohydroxamic acid: structure of the inert complex and its mechanistic interpretation.
Inhibition of chymotrypsin by a complex of ortho-vanadate and benzohydroxamic acid: structure of the inert complex and its mechanistic interpretation.
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DOI：
10.1021/bi6025209
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发表时间：
2007-05
期刊：
影响因子：
2.9
通讯作者：
A. Moulin;J. Bell;R. Pratt;D. Ringe
中科院分区：
文献类型：
--
作者：
A. Moulin;J. Bell;R. Pratt;D. Ringe
丝氨酸蛋白酶，如丝氨酸β-内酰胺酶，被适当设计的磷酸盐快速且共价地抑制。因此，这些酶的活性位点必须能够稳定磷基转移反应的五配位过渡态以及酰基转移的四面体过渡态。因此，这些酶也应该被能够产生惰性五配位物种的分子抑制。我们（J.H.B.和R. F. P.）事实上，这些酶被钒酸盐和异羟肟酸的1：1复合物快速且可逆地抑制。在本文中，我们提出的第一个晶体结构的酰基转移酶抑制钒酸盐。钒酸盐和苯并异羟肟酸的复合物是α-糜蛋白酶的竞争性抑制剂，KI值为16 μ M。在结构中，在1.5 A的分辨率下获得，蛋白质在构象上与脱辅基酶略有不同。钒，在一个扭曲的八面体配体领域，是共价键合的活性位点丝氨酸氧基团。一个氧配体，推测阴离子，位于含氧阴离子孔。另一个是直接大致在酰基转移离去基团的方向，和第三个在S2位点的方向。异羟肟酸盐通过羟基氧与钒结合，并且更弱地通过羰基与钒结合，以形成五元螯合环。这种螯合作用的作用是将抑制剂的苯基基团置于重要的S1特异性位点。异羟肟酸氧被引导成远离Ser 195 Ogamma的直线，近似于磷基转移中离去基团的离开方向。整个复合物可以被看作是一个合理的模拟磷转移过渡态，其中离去基团延伸到S1网站。
Serine proteases, like serine beta-lactamases, are rapidly and covalently inhibited by suitably designed phosph(on)ates. The active sites of these enzymes must, therefore, be able to stabilize the pentacoordinated transition states of phosphyl transfer reactions as well as the tetrahedral transition states of acyl transfers. It follows that these enzymes should also be inhibited by molecules capable of generating inert pentacoordinated species. We (J.H.B. and R.F.P.) have previously shown that these enzymes are, in fact, rapidly and reversibly inhibited by 1:1 complexes of vanadate and hydroxamic acids. In this paper, we present the first crystal structure of an acyl transferase inhibited by vanadate. The complex of vanadate and benzohydroxamic acid is a competitive inhibitor of alpha-chymotrypsin with a KI value of 16 muM. In the structure, obtained at a resolution of 1.5 A, the protein is conformationally little different from the apoenzyme. The vanadium, in a distorted octahedral ligand field, is covalently bound to the active site serine oxygen group. One oxgen ligand, presumably anionic, is located in the oxyanion hole. Another is directed roughly in the direction of the acyl transfer leaving group, and a third in the direction of the S2 site. The hydroxamate is bound to vanadium through the hydroxyl oxygen and also, more weakly, through the carbonyl group, to form a five-membered chelate ring. The effect of this chelation is to place the phenyl group of the inhibitor into the important S1 specificity site. The hydroxamate oxygen is directed in line away from the Ser195 Ogamma, approximating the direction of departure of a leaving group in phosphyl transfer. The entire complex can be seen as a reasonable mimic of a phosphyl transfer transition state where the leaving group is extended into the S1 site.