The rate-limiting catalytic steps of hydroxymandelate synthase from Amycolatopsis orientalis

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of p-hydroxyphenylglycine, a recurrent substructure of polycyclic nonribosomal peptide antibiotics such as vancomycin. HMS has the same structural fold as and uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD) (4-hydroxyphenylpyruvate (HPP) and O(2)). Moreover, HMS catalyzes a very similar dioxygenation reaction to that of HPPD, adding the second oxygen atom to the benzylic position, rather than the aromatic C1 carbon of the substrate. The dissociation constant for HPP (59 microM) was measured under anaerobic conditions by titrating substrate with enzyme and monitoring the intensity of the weak (epsilon(475nm ) approximately 250 M(-1) cm(-1)) charge-transfer absorption band of the HMS.Fe(II).HPP complex. Pre-steady-state analysis indicates that evidence exists for the accumulation of three intermediates in a single turnover and the decay of the third is rate-limiting in multiple turnovers. The rate constants used to fit the data were k(1) = 1 x 10(5) M(-1) s(-1), k(2) = 250 s(-1), k(3) = 5 s(-1), and k(4) = 0.3 s(-1). However, the values for k(1) and k(2) could not be accurately measured due to both a prolonged mixing time for the HMS system that obscures observation at the early times (<10 ms) and the apparent high relative value of k(2). The third phase, k(3), is attributed to the formation of the product complex, and no kinetic isotope effect was observed on this step when the protons of the substrate's benzylic carbon were substituted with deuteriums, suggesting that hydroxylation is fast relative to the steps observed. The final and predominantly rate-limiting step shows a 3-fold decrease in the magnitude of the rate constant in deuterium oxide solvent, and a proton inventory for this step suggests the contribution of a single proton from the solvent environment.