Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

Abstract

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evolution for nitrogenase proteins of K. pneumoniae, were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant Ki = 20 .mu.M was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K. pneumoniae occurred with a rate comparable with that of electron transfer, k .apprx. 2 .times. 102s-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data were fitted to symmetry and sequential-type models involving conformation changes in 2 identical subunits. The enzyme can bind up to 2 molecules of MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.