Bell Jason H, Pratt R F
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Biochemistry. 2002 Apr 2;41(13):4329-38. doi: 10.1021/bi012096v.
The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.
阴沟肠杆菌P99的C类β-内酰胺酶受到低浓度钒酸盐与异羟肟酸1:1复合物的竞争性抑制。构效关系研究表明，异羟肟酸官能团对这种抑制作用至关重要。芳基和烷基异羟肟酸均能与钒酸盐和酶形成抑制性三元复合物，不过，对于后者的某些情况，由于钒酸盐-异羟肟酸复合物的形成常数较低，可能观察不到抑制作用。在考虑了溶液中存在的所有钒酸盐物种后，可确定钒酸盐复合物的“真实”K(i)值。所研究的最佳抑制剂，即钒酸盐与4-硝基苯异羟肟酸的1:1复合物，其K(i)值为0.48微摩尔。动力学研究表明，该复合物与酶的缔合和解离速率常数分别为1.48×10(6) s(-1) M(-1)和0.73 s(-1)；后者的大小表明复合物与酶存在共价相互作用。(51)V核磁共振和紫外可见光谱表明，与酶结合的钒酸盐复合物的结构可能与溶液中的非常相似。酶与4-硝基苯[(13)C]异羟肟酸和钒酸盐的复合物的(13)C核磁共振谱产生了7.74 ppm的配位诱导位移（CIS）。这明显大于游离溶液中钒酸盐复合物的位移（3.62 ppm），这表明，与(51)V和紫外可见光谱有些相反，酶复合物中钒与异羟肟酸羰基氧之间的相互作用比游离溶液中更强，或者更有可能的是，通过与酶的相互作用（例如氢键）使异羟肟酸发生极化。分子模拟表明，五配位钒酸盐复合物很可能能够紧密占据酶的活性位点；天冬酰胺152的位置适合与异羟肟酸氧原子形成氢键。实验结果与一个模型相符，即钒酸盐-异羟肟酸-酶复合物是β-内酰胺酶与膦酸盐抑制剂反应过渡态的一个中等良好的类似物。
