Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis.
Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 +/- 0.2, 1.8 +/- 0.2, and 1.1 +/- 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
James D
JD
Garrity
Anne L
AL
Carenbauer
Lissa R
LR
Herron
Michael W
MW
Crowder
D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D011522:Protons; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry*
D008670:Metals; D011522:Protons; D001224:Aspartic Acid; C088032:beta-lactamase L1; D001618:beta-Lactamases; D015032:Zinc