Kinetic Studies on Hydrogenase

The kinetics of hydrogenase from Clostridium pasteurianum and from Proteus vulgaris were studied by measuring the HD and D/sub 2/ produced when the enzyme catalyzes the exchange between H/sub 2/ and 100% D/sub 2/O. The ratio of D/sub 2/ to HD production depends on the enzyme concentration because the HD produced by the exchange of H/sub 2/ can react with another enzyme molecule to give D/sub 2/ before it enters the gas phase. At very iow enzyme concentrations the ratio D/sub 2//HD is independent of the enzyme concentration. The D/sub 2// HD ratio depends on pH. For the hydrogenase from Clostridium pasteurianum this ratio is a maximum at a pH of 7.5, whereas the maximum in the enzyme activity occurs at a pH of 8.3. The rate of conversion of p- to o-hydrogen in pure D/sub 2/O was measured and found to be less than 1/10 the rate of the exchange reaction. The ratio D/sub 2/ to HD production and the rate of conversion of p-hydrogen are calculated for three types of activation of the H/sub 2/: a homolytic cleavage of the H/sub 2/ into two hydrogen atoms bound to the enzyme; a cleavage of the H/sub 2/ into anmore » enzyme hydride and a proton, with the hydride not exchanging with the solvent; and, a cleavage of the H/sub 2/ into an enzyme hydride and a proton, but with the hydride able to exchange with the solvent. The experimental data are consistent with the cleavage of the H/sub 2/ into a proton and an enzyme hydride that can exchange with the solvent. It is also possible to interpret these data in terms of a hydride that does not exchange, if it is assumed that there is a cage of water molecules or protein around the active site which allows the H/sub 2/ molecule to react several times before it leaves the vicinity of the enzyme. (auth)« less