PH and kinetic isotope effects on the oxidative half-reaction of D-amino-acid oxidase.

J.M Denu,P.F Fitzpatrick

doi:10.1016/s0021-9258(17)36573-0

Abstract

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.

Highlights

From the Departments of $Biochemistry a n d Biophysics a n d VChemistry, Texas A & M University, College Station, Texas 77843
The protoninventory at high pH is about the details of the oxidative half-reaction, we describe linear, consistent with release of a single proton in the here a mechanistic study of the oxidative half-reaction of D
Solvent Isotope Effects-Buffer components were added directly to D,O, and the pD was adjusted with NaOD or acetic acid-d,; pD values were determined by adding 0.4to the pH electrodereading (Schowenand Schowen,1982).The reaction was startedby the addition ofnomore than 10pl of enzyme in H,O to a final volume of 3 ml

Summary

Lactate dehydrogenaseand NADH were present to convert the product

Dept. of Biological Chemistry,University of Michigan, Ann Arbor,MI 48109. 11 Established investigator of the American Heart Association. D-AmOinxoid-acseid both varied, or the amino acid concentration was fixed at greater than 10-foldhigher than the K,,, value and only the oxygen concentrationwas varied. Solvent Isotope Effects-Buffer components were added directly to D,O, and the pD was adjusted with NaOD or acetic acid-d,; pD values were determined by adding 0.4to the pH electrodereading (Schowenand Schowen,1982).The reaction was startedby the addition ofnomore than 10pl of enzyme in H,O to a final volume of 3 ml. At pH 10.1 (pD = 10.6), 3 pl of a 3 5 - p D~ -amino-acid oxidasesolutionwere added to a 2.5-ml solutionof 50 mM D-alanine, 1.27 mM oxygen (KO,= 0.3 mM), and 50 mM ethanolamine, 25 "C. The observed rates of flavin reduction at various substrate concentrations were fit to Equation 1. If rates decreased only at high pH, the data were fit t o Equation 7. Equation 8 describes a linearproton inventory where n is the atom fraction of D,O, V, is the observed V, value at some n, and V, is the value in H,O

RESULTS

DISCUSSION

Proton inventories were measuredto elucidate the numberof