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

Abstract

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) 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 αKG 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.

Low pH Facilitates Heterodimerization of Mutant Isocitrate Dehydrogenase IDH1-R132H and Promotes Production of 2-Hydroxyglutarate

“…These structural data are supported by our prior collaborative work using computational analyses to identify a pH sensitive network of ionizable residues at the homodimerization interface of WT IDH1. 14 Our heterodimer data suggest that maintaining this hydrogen bond network via a positively charged Arg interacting with a negatively charged Asp is important for dimerization. Indeed, the increased heterodimer formation we observe specifically at low pHi, where His132 is more likely to be protonated, supports this hypothesis.…”

“… 13 Prior work suggests that WT IDH1 reactions could be sensitive to environment or microenvironment changes, with low buffer pH enhancing the reverse reaction (AKG → ICT) 13 and high buffer pH enhancing the forward reaction (ICT → AKG) in vitro . 14 These in vitro results with recombinant enzyme have some dependence on the specific buffer systems used, and the most pronounced effects require pH values outside the physiological range (7.0–7.6). However, this work does suggest that IDH1 function may be linked to environmental cues.…”

Probing altered enzyme activity in the biochemical characterization of cancer

“…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 ( Figure 2 ) [ 102 ].…”

Isocitrate dehydrogenase gene variants in cancer and their clinical significance

“…of isocitrate to 2OG and CO 2 , is usually studied at neutral pH, with the reverse reductive decarboxylation reaction being preferred at acidic pH [ 19 ]. The k cat for wtIDH catalysed isocitrate oxidation increases from 20.0 ± 0.4 s −1 to 38.3 ± 0.9 s −1 as the pH increases from pH 6.2 to pH 8 with maximal efficiency about pH 7.5, (1.4 ± 0.1) × 10 3 mM − 1 s − 1 [ 92 ]. These observations suggest that cellular pH may influence wtIDH/IDH variant catalysis.…”

“…Modulation of the ionisation state of Asp273 is proposed to be involved in the pH-mediated regulation of wtIDH; substitutions of Asp273 reduce catalytic efficiency and cause loss of pH regulatory effects [ 92 ]. The intracellular pH (pHi) has been measured as 7.01 ± 0.2 in normal brain in vivo by 31 P Magnetic resonance spectroscopy (MRS) but is more basic (pHi 7.18) in glioma [ 32 , 93 ].…”