Influence of substrates and coenzymes on the role of manganous ion in reactions catalyzed by pig heart triphosphopyridine nucleotide-dependent isocitrate dehydrogenase

Abstract

The interaction of manganous ions with pig heart triphosphopyridine nucleotide (TPN) specific isocitrate dehydrogenase has been studied by kinetic experiments and by direct ultrafiltration measurements of manganous ion binding. At low metal ion concentrations, a lag is observed in the time-dependent production of reduced triphosphopyridine nucleotide (TPNH) that can be eliminated by adding 20 muM TPNH to the initial reaction mixture. A plot of 1/upsilon vs. 1/ (Mn2+) obtained at relatively high TPNH concentrations (20 muM) is linear and yields of Km value of 2 muM for metal ion, which is comparable to the direct binding constant measured in the presence of isocitrate. A similar plot at low TPNH concentrations (2 muM) reveals a biphasic relationship: at high metal concentrations the points are collinear with those obtained at high levels of TPNH, but at low metal concentrations that line is characterized by a Km of 19 muM for Mn2+. A difference in the deuterium oxide solvent isotope effect on Vmax observed with 20 muM TPNH as compared with 2 muM TPNH suggests that at high TPNH concentrations or high manganous ion concentrations the rate-limiting step is the dehydrogenation of isocitrate, while at low manganous ion concentrations and low TPNH concentrations, the slow step is the decarboxylation of enzyme-bound oxalosuccinate. Evidence to support this hypothesis is provided by the sensitivity to isocitrate concentration of the Km for total manganese measured in the presence of 20 muM TPNH that contrasts with the relative insensitivity to isocitrate of the Km measured at 2 muM TPNH and low manganous ion concentration. Direct measurements of oxalosuccinate decarboxylation reveal that the Vmax and the Km for manganous ion are influenced by the presence of oxidized or reduced TPN with the Km being lowest (5-7 muM) in the presence of TPNH. The dependence of the Km for manganous ion on the presence of substrate, TPN, and TPNH, is responsible for the variation with conditions in the rate-determining step. The enzyme binds only 1 mol of metal ion and 1 mol of isocitrate/mol of protein under all conditions. The pH dependence of the binding of free manganous ion, free isocitrate, and manganous-isocitrate complex indicates differences in the interaction of these species with isocitrate dehydrogenase. These results can be described in terms of two functions for manganous ion in the reactions catalyzed by isocitrate dehydrogenase, each of which requires a distinct binding site for metal ion: in the dehydrogenation step, Mn2+ facilitates the binding of the substrate isocitrate, and in the decarboxylation step it may stabilize the enolate of alpha-ketoglutarate which is generated.

The Common Feature of Leukemia-Associated IDH1 and IDH2 Mutations Is a Neomorphic Enzyme Activity Converting α-Ketoglutarate to 2-Hydroxyglutarate

“…In IDH1, the role of R132 in determining IDH1 enzymatic activity is consistent with the stabilizing charge interaction of its guanidinium moiety with the β-carboxyl group of isocitrate (Figure 1A). This β-carboxyl is critical for IDH’s ability to catalyze the interconversion of isocitrate and α-ketoglutarate, with the overall reaction occurring in two steps through a β-carboxyl-containing intermediate (Ehrlich and Colman, 1976). Proceeding in the oxidative direction, this β-carboxyl remains on the substrate throughout the IDH reaction until the final decarboxylating step which produces α-ketoglutarate.…”

Transient kinetics of nicotinamide–adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria

“…There have also been many previous reports of time lags from steady-state kinetic studies of NADPlinked isocitrate dehydrogenase from various sources, including bovine heart (Londesborough, 1969), pig heart (Sanner & Ingebretsen, 1976;Ingebretsen & Sanner, 1976;Ehrlich & Colman, 1976) and pig liver (Carlier & Pantaloni, 1976). Again no correlation with the effects that we describe is apparent; the time lags reported by these earlier workers were generally more prolonged, or were observed under different conditions, notably in phosphate buffer and with very small concentrations of metal activator, and were abolished by NADPH in some cases.…”

Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP‐dependent isocitrate dehydrogenase

“…In contrast, substitution of alanine for Ser 95 or Thr 78 results in about a 10-fold increase in the K m for isocitrate, and the effects on this K m are even greater in S95D and T78D. There are corresponding increases in the K m for Mn 2+ , as might be expected since Mn 2+ -isocitrate is considered to be the preferred substrate for the NADP-dependent isocitrate dehydrogenase (Colman 1972(Colman , 1983Villafranca and Colman 1972;Ehrlich and Colman 1976).…”