The autocatalytic step is an integral part of the hydrogenase cycle

We earlier proved the involvement of an autocatalytic step in the oxidation of H-2 by Hyna, hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H-2, we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme-substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment. (C) 2013 Elsevier B.V. All rights reserved.