Kinetic Mechanism Studies of the Soluble Hydrogenase from Alcaligenes EutrophusH161

摘要

Purified soluble hydrogenase (112:NAD+ oxidoreductase, EC 1.12.1.2) fromAlcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH. 112-dependent NAD+ reduction activities> 110 %mol NADH formed/min/mg protein at pH 8.0 and 300 C were obtained which were stable for several hours at 40% 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 the hyperbaric conditions (i.e., at ambient pressures greater than 1 atm). Using nonlinear curve fitting of the kinetic data, it was found that 112 and NAD+ bind hydrogenase via a ping pong bi