Site-directed mutagenesis of histidine 238 in mouse adenosine deaminase: substitution of histidine 238 does not impede hydroxylate formation

Biochemistry
V SiderakiF B Rudolph

Abstract

His 238, a conserved amino acid located in hydrogen-bonding distance from C-6 of the substrate in the active site of murine adenosine deaminase (mADA) and postulated to play an important role in catalysis, was altered into an alanine, a glutamate, and an arginine using site-directed mutagenesis. The Ala and Glu substitutions did not result in changes of the secondary or tertiary structure, while the Arg mutation caused local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans. Neither the Glu or Arg mutations affected substrate binding affinity. By contrast, the Ala mutation enhanced substrate and inhibitor binding by 20-fold. The most inactive of the mutants, Glu 238, had a kcat/K(m) 4 x 10(-6) lower than the wild-type value, suggesting that a positive charge on His 238 is important for proper catalytic function. The Ala 238 mutant was the most active ADA, with a kcat/K(m) 2 x 10(-3) lower than the wild-type value. NMR spectroscopy and crystallography revealed that this mutant is able to catalyze hydration of purine riboside, a ground-state analog of the reaction. These results collectively show that His 238 is not required for formation of the hydroxylate used in the deamination and...Continue Reading

Citations

Feb 27, 2001·Medicinal Research Reviews·G CristalliE Camaioni
Oct 26, 2005·Journal of Molecular Evolution·Stephanie A MaierHeather E McDermid
Jun 30, 2011·Biochemistry·Alissa M GobleFrank M Raushel
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Jun 25, 2010·Journal of Computational Chemistry·Xian-Hui WuYun-Dong Wu
Jun 17, 2014·Medicinal Research Reviews·Antoni CortésVicent Casadó
Oct 16, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Barbara Kutryb-ZajacRyszard T Smolenski
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