Probing the role of Asp-120(81) of metallo-β-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography

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 pKa 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 angstrom resolutions, respectively. In the case of D120(81) E, the Zn-Zn distance was increased by 0.3 angstrom 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.