Stopped-flow spectrophotometric studies on the reaction of turkey liver xanthine dehydrogenase with reducing substrates.

The kinetics of reduction of turkey liver xanthine dehydrogenase by substrates were investigated by stopped-flow spectrophotometry. The results may be explained in terms of the known redox potentials of the various centres in the enzyme [Barber, Bray, Cammack & Coughlan (1977) Biochem. J. 163, 279-289]. They are, morover, consistent with the scheme [Olson, Ballou, Palmer & Massey (1974) J. Biol. Chem. 249, 4363-4382] in which reduction occurs in three consecutive steps, one molecule of substrate reacting with the active site at each step. First-order rate constants believed to correspond respectively to the combined first and second steps and to the third step in the reduction by excess of xanthine and of NADH were determined. The rates of reaction with these substrates in the combined first and second steps are independent of the degree of enzyme functionality.