Transient State Analysis of Human Isocitrate Dehydrogenase I: Accounting for the Interconversion of Active and Non-Active Conformational States.

Human isocitrate dehydrogenase 1 (HsICDH1) is a cytoplasmic homodimeric Mg(II)-dependent enzyme that converts D-isocitrate (D-ICT) and NADP+ to alpha-ketoglutarate (AKG), CO2 and NADPH. The active sites are formed at the subunit interface and incorporate residues from both protomers. The turnover number titrates hyperbolically from 17.5 s-1 to a minimum of 7 s-1 with increasing enzyme concentration. As isolated, the enzyme adopts an inactive open conformation and binds NADPH tightly. The open conformation displaces three of the eight residues that bind D-ICT and Mg(II). Enzyme activation occurs with the addition of Mg(II) or D-ICT with a rate constant of 0.12 s-1. The addition of both Mg(II) and D-ICT activates the enzyme with a rate constant of 0.6 s-1 and displaces half of the bound NADPH. This indicates that HsICDH1 utilizes a half-site mechanism in which the active sites alternate in catalysis. The X-ray crystal structure of the half-site activated complex reveals asymmetry in the homodimer with a sin...