Pre-steady-state kinetic studies on the Glu171Gln active site mutant of adenosylcobalamin-dependent glutamate mutase.
Glutamate-171 is involved in recognizing the amino group of the substrate in glutamate mutase. The effect of mutating this residue to glutamine on the ability of the enzyme to catalyze the homolysis of adenosylcobalamin has been investigated using UV-visible stopped-flow spectroscopy. Although Glu171 does not contact the coenzyme, the mutation results in the apparent rate constants for substrate-induced homolysis of the coenzyme that are slower by 7-fold and 13-fold with glutamate and methylaspartate, respectively, than those measured for the wild-type enzyme; furthermore, it weakens the binding of these substrates by approximately 50-fold and approximately 400-fold, respectively. These observations lend support to the idea that the enzyme may use substrate binding energy to accelerate homolysis of the coenzyme. The mutation also results in isotope effects on coenzyme homolysis that are much smaller than the very large effects observed when the wild-type enzyme is reacted with deuterated substrates. This observation is consistent with adenosylcobalamin homolysis being slowed relative to hydrogen abstraction from the substrate.
Madhavapeddi
Prashanti
P
Department of Chemistry, Division of Biophysics, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Ballou
David P
DP
Marsh
E Neil G
EN
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003038:Cobamides / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics*; D005973:Glutamine / Q000235:genetics; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016202:N-Methylaspartate / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000235:genetics
D003038:Cobamides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D016202:N-Methylaspartate; D003903:Deuterium; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide