Yamaguchi Yoshihiro, Kuroki Takahiro, Yasuzawa Hisami, Higashi Toshihiro, Jin Wanchun, Kawanami Akiko, Yamagata Yuriko, Arakawa Yoshichika, Goto Masafumi, Kurosaki Hiromasa
Department of Structure-Function Physical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan.
J Biol Chem. 2005 May 27;280(21):20824-32. doi: 10.1074/jbc.M414314200. Epub 2005 Mar 23.
Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.
金属β-内酰胺酶IMP-1是一种双锌（II）金属酶，能高效水解β-内酰胺类抗生素。野生型（WT）IMP-1在活性位点有一个保守的天冬氨酸-120（81），它在催化过程中起重要作用。为了探究天冬氨酸-120（81）在IMP-1中的催化作用，通过定点诱变制备了IMP-1突变体D120（81）A和D120（81）E，并进行了各种动力学研究。尽管IMP-1突变体在活性位点含有两个锌（II）离子，但与野生型相比，它们的催化常数（kcat）值下降了10²-10⁴倍。为了评估天冬氨酸-120（81）的酸碱特性，通过停流研究考察了水解反应对pH的依赖性。对于野生型和D120（81）A，在pH 5至9之间未发现可观察到的pKa值。等摩尔量的硝噻吩和所有酶快速混合，未能在650 nm处检测到硝噻吩的阴离子中间体。这些结果表明，IMP-1的天冬氨酸-120（81）不是降低两个锌（II）离子之间水的pKa的因素，也不是阴离子中间体的质子供体。在D120（81）E的情况下，在460 nm处显示最大吸收的硝噻吩水解产物以质子化形式与D120（81）E结合。D120（81）A和D120（81）E的三维结构也分别在2.0和3.0埃分辨率下确定。在D120（81）E的情况下，由于活性位点处谷氨酸-120（81）OE1的配位模式变化以及保守的组氨酸-263（197）的位置移动，锌-锌距离比野生型增加了0.3埃。
