An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Abstract

§This paper is dedicated to the memory of our dear colleague and friend, Michelle Evon Scott (1990Scott ( -2020. ABSTRACTIsocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (α-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for α-KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ~12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Probing altered enzyme activity in the biochemical characterization of cancer

“…This pH sensitivity can often be driven by the presence of buried ionizable residues such as arginine, lysine, histidine, aspartic acid, or glutamic acid if the residue's pKa value is in a physiologically relevant range, allowing it to sense small changes in pH (for cancer cells, usually in the range of pH 6.7-7.6) [97][98][99]. Proteins that detect and respond to changes in pH by altering activity are known as pH sensors [100], with examples including members of ATPases, GTPases, kinases, and metabolic enzyme families [101][102][103][104][105].…”

“…Recently, we described a mechanism of pH regulation of WT IDH1 catalysis, a long-described phenomenon whose mechanism was not well understood. We found that the catalytic rate of the forward reaction (isocitrate to αKG) was increased upon increasing pH [102]. To establish the mechanism of pH sensitivity, we identified a buried aspartic acid residue in WT IDH1, D273, that sensed local changes in pH likely by undergoing a change in protonation state (Fig.…”

“…To establish the mechanism of pH sensitivity, we identified a buried aspartic acid residue in WT IDH1, D273, that sensed local changes in pH likely by undergoing a change in protonation state (Fig. 2) [102]. This residue is found in the 10 regulatory domain, which undergoes a conformational change to restructure the active site from an inhibitory to a catalytically competent form (Fig.…”