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 pK(a) value of about 10.5 be protonated and a group with a pK(a) 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/K(O₂) 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 V(max) 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 (D)V 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.