6PAY

Structure of HsICDH1:Mg(II):ICT:NADPH(50%) complex reveals structural basis for observation of half-sites reactivity

Summary for 6PAY

• Entry DOI: 10.2210/pdb6pay/pdb
• Descriptor: Isocitrate dehydrogenase [NADP] cytoplasmic, ISOCITRIC ACID, MAGNESIUM ION, ... (6 entities in total)
• Functional Keywords: substrate complex, half-sites reactivity, icdh mechanism, oxidoreductase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 4
• Total formula weight: 188814.20

Authors

Silvaggi, N.R.,Melkonian, T.R.,Roman, J.V.,Moran, G.R. (deposition date: 2019-06-12, release date: 2019-09-18, Last modification date: 2023-10-11)

Primary citation

Roman, J.V.,Melkonian, T.R.,Silvaggi, N.R.,Moran, G.R.
Transient-State Analysis of Human Isocitrate Dehydrogenase I: Accounting for the Interconversion of Active and Non-Active Conformational States.
Biochemistry, 58:5366-5380, 2019

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, 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 to a minimum of 7 s 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. The addition of both Mg(II) and D-ICT activates the enzyme with a rate constant of 0.6 s 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 followed by a shift in the NADPH absorption spectrum with a rate constant of 28 s. 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.

PubMed: 31478653
DOI: 10.1021/acs.biochem.9b00518

Experimental method

X-RAY DIFFRACTION (2.199 Å)