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J. Biol. Chem., Vol. 279, Issue 2, 920-927, January 9, 2004

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Metal Binding Asp-120 in Metallo--lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis^*

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

From the Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056

Metallo--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 -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 k_cat 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 pK_a 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.

Received for publication, September 4, 2003 , and in revised form, October 20, 2003.

^* This work was supported by National Institutes of Health Grants AI40052 and GM40052. Funds to purchase the CD spectrapolarimeter (DBI-0070169) and the stopped-flow UV-visible fluorescence spectrophotometer (CHE-0076936) were provided by the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, OH 45056. Tel.: 513-529-7274; Fax: 513-529-5715; e-mail: crowdemw{at}muohio.edu.

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