Kinetic Isotope Effects as Probes of the Mechanism of Galactose Oxidase (1998)

Galactose oxidase (GO) is a member of the family of radical-coupled copper oxidases, enzymes containing a free radical coordinated to copper in the active site. In catalysis GO cycles between an oxidized state (comprising Cu(II) with a unique cysteinyl-tyrosine radical) and a reduced state (comprising Cu(I) with the singlet cysteinyl-tyrosine) as it catalyzes the two-electron oxidation of alcohols to aldehydes and the subsequent reduction of O2 to H2O2. A ping-pong mechanism involving radical intermediates has been proposed for GO catalysis. Previous steady-state kinetics studies have demonstrated a KIE of 7-8 that was attributed to substrate oxidation, a process involving the stereospecific abstraction of the pro-S hydrogen from the 6-hydroxymethyl group of galactose. We have used rapid kinetics methods to measure the anaerobic reduction of GO substrate at 4 degreesC and carry out enzyme-monitored turnover experiments using 6-protio and 6-deutero substrates, both in H2O and D2O. At concentrations below Km, the apparent second-order rate constant for protio-substrate oxidation, kred, was 1.59 x 10(4) M-1 s-1, while that for deuterated substrate was 7.50 x 10(2) M-1 s-1, a KIE of 21.2. Steady-state measurements of oxygen consumption at low galactose concentrations reveal an unusually large isotope effect (kH/kD = 22.5 +/- 2) for oxidation of 1-O-methyl-6, 6'-di-[2H]-alpha-d-galactopyranoside, and at high galactose concentrations, where the oxygen half-reaction is rate-limiting in catalysis, a surprisingly large KIE (kH/kD = 8 +/- 1) for the reduction of O2 to H2O2. There is no detectable solvent isotope effect (<5%) on any of these measurements. This shows that there are no exchangeable protons involved in any kinetically significant step and that the hydrogen atom removed from galactose is not lost to solvent during catalysis; instead, it also participates in the rate-limiting step of the subsequent reaction with oxygen. At concentrations below Km, apparent second-order rate constants for protio-substrate oxidation (kred = 1.5 x 10(4) M-1 s-1) and O2 reduction (kox = 8 x 10(6) M-1 s-1) have been estimated from measurements both by steady-state oxygen electrode and by enzyme-monitored turnover. This is completely consistent with the anaerobic studies mentioned above. Our results show that the enzyme is essentially fully oxidized while in steady-state turnover, consistent with the reduction step being nearly fully rate-limiting at practical substrate concentrations, due to the very fast reaction with physiological concentrations of O2. Overall, the catalytic reaction is in concordance with a ping-pong mechanism. The large KIE associated with reduction of the enzyme in all three methods appears to reflect hydrogen atom radical abstraction by the active site tyrosine radical in the rate-determining step, in agreement with the previously proposed radical mechanism for GO. The KIE determined at low substrate concentrations (where oxidation of substrate is rate determining) from steady-state oxygen consumption measurements, varies from 22.5 at 4 degreesC to 13 at 45 degreesC, consistent with tunneling being involved in the hydrogen atom transfer step.