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Structure of HsICDH1:Mg(II):ICT:NADPH(50%) complex reveals structural basis for observation of half-sites reactivity
Structural highlights
Disease[IDHC_HUMAN] Defects in IDH1 are involved in the development of glioma (GLM) [MIM:137800]. Gliomas are central nervous system neoplasms derived from glial cells and comprise astrocytomas, glioblastoma multiforme, oligodendrogliomas, and ependymomas. Note=Mutations affecting Arg-132 are tissue-specific, and suggest that this residue plays a unique role in the development of high-grade gliomas. Mutations of Arg-132 to Cys, His, Leu or Ser abolish magnesium binding and abolish the conversion of isocitrate to alpha-ketoglutarate. Instead, alpha-ketoglutarate is converted to R(-)-2-hydroxyglutarate. Elevated levels of R(-)-2-hydroxyglutarate are correlated with an elevated risk of malignant brain tumors. Publication Abstract from PubMedHuman 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 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 approximately 3 s(-1). Transient-State Analysis of Human Isocitrate Dehydrogenase I: Accounting for the Interconversion of Active and Non-Active Conformational States.,Roman JV, Melkonian TR, Silvaggi NR, Moran GR Biochemistry. 2019 Dec 31;58(52):5366-5380. doi: 10.1021/acs.biochem.9b00518., Epub 2019 Sep 10. PMID:31478653[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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