Kinetic Mechanism Studies of the Soluble Hydrogenase from Alcaligenes eutrophus H16

Purified soluble hydrogenase (H2:NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH. H2-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 H2/O2 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 H2 and NAD+ bind hydrogenase via a ping pong bi bi mechanism with Km 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 H2 occurs at high substrate concentrations (Ki = 1.46 ± 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H2.