The kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase have been studied using the physiological substrates pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) at 25 degrees C and pH 8.0. Under steady-state conditions with different concentrations of PNP and O2, a series of parallel lines and competitive substrate inhibition with a KI of 50 microM are seen in double reciprocal plots. This is suggestive of a binary complex mechanism. Secondary plots yield a turnover number of 42 min-1 and Km values for both PNP (8.2 microM) and O2 (182 microM). A large deuterium isotope effect, VH/VD of 6.5, was observed with [4',4'-2H]PNP. In analogous studies using PMP, a turnover number of 6.2 min-1 and respective Km values for PMP and O2 of 3.6 and 85 microM were calculated. No significant substrate inhibition and a small deuterium isotope effect (VH/VD = 1.1) were observed with PMP. Anaerobic stopped flow data showed that the enzyme-bound flavin was reduced at a rate similar to catalytic turnover with PNP as a substrate, whereas with PMP, the rate of enzyme reduction is 500-fold faster than turnover. Stopped flow kinetic data also showed the reduced enzyme to react with O2 at rates at least 10(2)-10(3) faster than turnover. These results indicate that enzyme reduction is rate-limiting when the alcohol form (PNP) is the substrate, but in the case of the amine (PMP), the rate-limiting step in catalysis occurs subsequent to reduction. With PMP as substrate, release of product from the complex with reduced enzyme is 15-fold slower than turnover, and thus, it is suggested that oxygen reacts with the complex. The pH dependence of the deuterium isotope effect and the Km for PMP showed substantial change in the pH range between 6.0 and 7.5, whereas little or no pH dependence was observed for PNP. These data show that the kinetic mechanism of pyridoxamine (pyridoxine)-5'-phosphate oxidase can function via either a binary or ternary complex mechanism, depending upon the nature of the substrate.