Role of Arg100 in the active site of adenosylcobalamin-dependent glutamate mutase.
Arginine-100 is involved in recognizing the gamma carboxylate of the substrate in glutamate mutase. To investigate its role in substrate binding and catalysis, this residue was mutated to lysine, tyrosine, and methionine. The effect of these mutations was to reduce k(cat) by 120-320-fold and to increase K(m(apparent)) for glutamate by 13-22-fold; K(m(apparent)) for adenosylcobalamin is little changed by these mutations. Even at saturating substrate concentrations, no cob(II)alamin could be detected in the UV-visible spectra of the Arg100Tyr and Arg100Met mutants. However, in the Arg100Lys mutant cob(II)alamin accumulated to concentrations similar to wild-type enzyme, which allowed the pre-steady-state kinetics of adenosylcobalamin homolysis to be investigated by stopped-flow spectroscopy. It was found that homolysis of the coenzyme is slower by an order of magnitude, compared with wild-type enzyme. Furthermore, glutamate binding is significantly weakened, so much so that the reaction exhibits second-order kinetics over the range of substrate concentrations used. The Arg100Lys mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild-type enzyme is reacted with deuterated substrates; this suggests that homolysis is slowed relative to hydrogen abstraction by this mutation.
Xia
Li
L
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Ballou
David P
DP
Marsh
E Neil G
EN
D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003038:Cobamides / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006860:Hydrogen Bonding; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016202:N-Methylaspartate / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity / Q000235:genetics; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000737:chemistry
D001426:Bacterial Proteins; D003038:Cobamides; C097751:cob(II)alamin; D018698:Glutamic Acid; D016202:N-Methylaspartate; D001120:Arginine; D003903:Deuterium; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide; D008239:Lysine; D014805:Vitamin B 12