pH and kinetic isotope effects on the oxidative half-reaction of D-amino-acid oxidase.
D-Amino-acid oxidase catalyzes the oxidation of D-amino acids to imino acids. In the oxidative half-reaction, oxygen reacts with the reduced enzyme-imino acid complex to reoxidize the bound FAD. This is then followed by dissociation of the imino acid. The effects of pH and D2O on the kinetics of the oxidative half-reaction of D-amino-acid oxidase have been determined with glycine, D-alanine, and D-serine as substrates. Reaction of the reduced enzyme with oxygen requires that a group with a pKa value of about 10.5 be protonated and a group with a pKa value of 8.5 be deprotonated. The former value is not seen with D-alanine as substrate; the latter is only seen with glycine. No solvent isotope effects are seen on the V/KO2 value with D-alanine, consistent with rate-limiting electron transfer. Product release involves a pH-dependent conformational change. This is rate-limiting at all pH values with D-alanine as substrate. Significant solvent isotope effects are seen on the Vmax value with D-alanine. The proton inventory at high pH is linear, consistent with release of a single proton in the slow step; at pH 6 the solvent inventory is bowl-shaped, consistent with a solvent isotope effect on the conformation of the protein. With glycine the DV value increases to the intrinsic value at pH 10.5; this establishes that CH bond cleavage becomes rate-limiting with this substrate above pH 10.
Denu
J M
JM
Department of Biochemistry, Texas A & M University, College Station 77843.
Fitzpatrick
P F
PF
D000409:Alanine / Q000378:metabolism; D000818:Animals; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D010084:Oxidation-Reduction; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D007554:Isotopes; D012694:Serine; D003605:D-Amino-Acid Oxidase; D000409:Alanine; D005998:Glycine