Kinetic mechanism studies of the soluble hydrogenase from Alcaligenes eutrophus H16.

Abstract Purified soluble hydrogenase (H 2 :NAD + oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N 2 and H 2 and then adding substoichiometric quantities of NADH. H 2 -dependent NAD + reduction activities ≥ 110 μmol NADH formed/min/mg protein at pH 8.0 and 30°C were obtained which were stable for several hours at 4°C. Kinetic studies were conducted anaerobically using activated enzyme for the purpose of evaluating the potential of using hydrogenase to enhance decompression of mammals breathing H 2 /O 2 mixtures under hyperbaric conditions (i.e., at ambient pressures greater than 1 atm). Using nonlinear curve fitting of the kinetic data, it was found that H 2 and NAD + bind hydrogenase via a ping pong bi bi mechanism with K m values (±SE) of 11 ± 0.9 and 138 ± 11 μM, respectively, at 30°C and pH 8.0. Sodium ions were found to reversibly inhibit hydrogenase via a dead-end type of inhibition in which two catalytic forms of the enzyme bind Na + with dissociation constants calculated to be 8.3 ± 1.2 and 49.8 ± 11.5 mM. In the absence of NaCl, maximum NAD + reduction activity was measured at pH 8.3 at 30 and 37°C. In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values ≤ 7.0, little or no difference was observed in activity in the presence or absence of 50 mM NaCl at a given temperature. Least squares analyses of the kinetic data indicated that substrate inhibition by H 2 occurs at high substrate concentrations ( K i = 1.46 ± 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H 2 .