SITE-DIRECTED MUTAGENESIS OF HISTIDINE-238 IN MOUSE ADENOSINE-DEAMINASE - SUBSTITUTION OF HISTIDINE 238 DOES NOT IMPEDE HYDROXYLATE FORMATION

His 238, a conserved amino acid located in hydrogen-bonding distance f rom C-6 of the substrate in the active site of murine adenosine deamin ase (mADA) and postulated to play an important role in catalysis, was altered into an alanine, a glutamate, and an arginine using site-direc ted mutagenesis. The Ala and Glu substitutions did not result in chang es of the secondary or tertiary structure, while the Arg mutation caus ed local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans. Neither the Glu or Arg mutations a ffected substrate binding affinity. By contrast, the Ala mutation enha nced substrate and inhibitor binding by 20-fold. The most inactive of the mutants, Glu 238, had a k(cat)/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 k(cat)/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 re action. These results collectively show that His 238 is not required f or formation of the hydroxylate used in the deamination and may instea d have an important electrostatic role.