Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis

James D Garrity, Anne L Carenbauer, Lissa Herron, Michael W Crowder

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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.
Original languageEnglish
Pages (from-to)920-7
Number of pages8
JournalJournal of Biological Chemistry
Issue number2
Publication statusPublished - Jan 2004

Keywords / Materials (for Non-textual outputs)

  • Aspartic Acid/chemistry
  • Binding Sites
  • Catalysis
  • Circular Dichroism
  • Escherichia coli/metabolism
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Kinetics
  • Metals/metabolism
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Folding
  • Protons
  • Stenotrophomonas maltophilia/enzymology
  • Zinc/chemistry
  • beta-Lactamases/chemistry


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