Metallo-beta-lactamase IMP-1 is a di-Zn(II) metallo-enzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type IMP-1 (WT) has a conserved Asp120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp120(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 10-10^4 fold drops in kcat compared to WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pKa’s 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 Asp120(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 Å and 3.0 Å resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 Å compared to WT, due to the change in the coordination mode of Glu120(81)OE1 and the positional shift in the conserved His263(197) at the active site.