Download MendeleyTransient-State Analysis of Human Isocitrate Dehydrogenase I: Accounting for the Interconversion of Active and Non-Active Conformational States.
Roman, J.V., Melkonian, T.R., Silvaggi, N.R., Moran, G.R.(2019) Biochemistry 58: 5366-5380
- PubMed: 31478653
- DOI: https://doi.org/10.1021/acs.biochem.9b00518
- Primary Citation of Related Structures:
6PAY - PubMed Abstract:
Human isocitrate dehydrogenase 1 (HsICDH1) is a cytoplasmic homodimeric Mg(II)-dependent enzyme that converts d-isocitrate (D-ICT) and NADP + to α-ketoglutarate (AKG), CO 2 , 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 an 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 may have 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 single NADPH bound. The structure also indicates a pseudotetramer interface that impedes the egress of NADPH consistent with the suppression of the turnover number at high enzyme concentrations. When the half-site activated form of the enzyme is reacted with NADP + , NADPH forms with a rate constant of 204 s -1 followed by a shift in the NADPH absorption spectrum with a rate constant of 28 s -1 . These data indicate the accumulation of two intermediate states. Once D-ICT is exhausted, HsICDH1 relaxes to the inactive open state with a rate constant of ～3 s -1 .
Organizational Affiliation:
Department of Chemistry and Biochemistry , Loyola University Chicago , Flanner Hall, 1068 West Sheridan Road , Chicago , Illinois 60660 , United States.
