Moulin Aaron, Bell Jason H, Pratt R F, Ringe Dagmar
Rosenstiel Basic Medical Sciences Research Center, Program in Biochemistry, and Program in Biophysics, Brandeis University, Waltham, Massachusetts 02454, USA.
Biochemistry. 2007 May 22;46(20):5982-90. doi: 10.1021/bi6025209. Epub 2007 May 1.
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.
丝氨酸蛋白酶与丝氨酸β-内酰胺酶一样，可被设计得当的磷酸（膦）酸酯快速且共价抑制。因此，这些酶的活性位点必须能够稳定磷酰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。由此可知，这些酶也应被能够生成惰性五配位物种的分子所抑制。我们（J.H.B.和R.F.P.）先前已表明，这些酶实际上会被钒酸盐与异羟肟酸的1:1复合物快速且可逆地抑制。在本文中，我们展示了首个被钒酸盐抑制的酰基转移酶的晶体结构。钒酸盐与苯甲酰异羟肟酸的复合物是α-胰凝乳蛋白酶的竞争性抑制剂，其抑制常数（KI）值为16 μM。在分辨率为1.5 Å时获得的该结构中，蛋白质的构象与脱辅基酶几乎没有差异。处于畸变八面体配体场中的钒与活性位点丝氨酸的氧原子共价结合。一个氧配体大概呈阴离子状态，位于氧负离子孔中。另一个大致指向酰基转移离去基团的方向，第三个指向S2位点的方向。异羟肟酸通过羟基氧与钒结合，同时也较弱地通过羰基与钒结合，形成一个五元螯合环。这种螯合作用的效果是将抑制剂的苯基置于重要的S1特异性位点中。异羟肟酸氧的方向与Ser195的Oγ方向成一条直线，近似于磷酰基转移中离去基团的离去方向。整个复合物可被视为磷酰基转移过渡态的合理模拟物，其中离去基团延伸至S1位点。
