title: Kinetic mechanism studies of the soluble hydrogenase from Alcaligenes eutrophus H16.
abstract: 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 > or = 110 mumol NADH formed/min/mg protein at pH 8.0 and 30 degrees C were obtained which were stable for several hours at 4 degrees 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 microM, respectively, at 30 degrees 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 degrees C. In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values < or = 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.
authors.affiliation: Naval Medical Research Institute, Bethesda, Maryland 20889-5607.
authors.forename: R G
authors.initials: RG
authors.lastname: Keefe
authors.forename: M J
authors.initials: MJ
authors.lastname: Axley
authors.forename: A L
authors.initials: AL
authors.lastname: Harabin
mesh_terms: D000421:Alcaligenes / Q000201:enzymology*; D000643:Ammonium Chloride / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D000643:Ammonium Chloride; D009243:NAD; D012965:Sodium Chloride; D011189:Potassium Chloride; D012964:Sodium; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase