Elucidation of the Role of Arginine-224 in the Turnover Processes of Class A β-Lactamases

Glenn Zafaralla, Elias K. Manavathu, Stephen A. Lerner, Shahriar Mobashery

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Abstract

The highly conserved arginine-244 of β-lactamases has been postulated to play a role in their initial recognition of substrates, presumably through ion pairing interactions [Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P., & Frere, J. M. (1990) Proteins·. Struct., Funct., Genet. 7, 156-171]. However, in the Michaelis enzyme-substrate complex, no direct function has been attributed to this residue. Two mutants with substitutions of this residue in the TEM-1 β-lactamase (lysine-244 and serine-244) have been prepared to explore whether the guanidinium group of arginine-244 plays a critical role in the turnover processes. The mutant enzymes are effective catalysts for the hydrolysis of both penicillins and cephalosporins, and the lysine mutant enzyme behaves virtually identically to the wild-type β-lactamase. Comparative kinetic characterization of the serine mutant and wild-type enzymes attributed apparent binding energies of 1.3-2.3 kcal/mol for the penicillins and 0.3-1.0 kcal/mol for the cephalosporins to the transition-state species by arginine-244. Furthermore, it was shown that arginine-244 also contributes equally well to ground-state binding stabilization. These results were interpreted to indicate the involvement of a long hydrogen bond between arginine-244 and the substrate carboxylate, both in the ground and transition states. A reassessed picture for substrate anchoring involving interactions of the substrate carboxylate with the side chains of Ser-130, Ser-235, and Arg-244 is proposed to accommodate these observations.

Original languageEnglish (US)
Pages (from-to)3847-3852
Number of pages6
JournalBiochemistry
Volume31
Issue number15
DOIs
StatePublished - Apr 1 1992

ASJC Scopus subject areas

  • Biochemistry

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    Zafaralla, G., Manavathu, E. K., Lerner, S. A., & Mobashery, S. (1992). Elucidation of the Role of Arginine-224 in the Turnover Processes of Class A β-Lactamases. Biochemistry, 31(15), 3847-3852. https://doi.org/10.1021/bi00130a016