Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis
| Autor: | Anne L. Carenbauer, Lissa R. Herron, Michael W. Crowder, James D. Garrity |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Circular dichroism Protein Folding Stereochemistry Stenotrophomonas maltophilia Biochemistry Catalysis beta-Lactamases Hydrolysis chemistry.chemical_compound Aspartic acid Escherichia coli Organic chemistry Enzyme kinetics Molecular Biology chemistry.chemical_classification Aspartic Acid Binding Sites Hydrogen bond Circular Dichroism Hydrogen Bonding Cell Biology Hydrogen-Ion Concentration Kinetics Zinc Enzyme chemistry Metals Mutation Mutagenesis Site-Directed Hydroxide Protons Protein Binding |
| Zdroj: | The Journal of biological chemistry. 279(2) |
| ISSN: | 0021-9258 |
| Popis: | 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-typeD120ND120C 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. |
| Databáze: | OpenAIRE |
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