Evidence from carbon-13 NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Abstract

The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

“…Polarization of the thioester carbonyl group has been observed by vibrational and NMR spectroscopies for a number of CoA-dependent enzymes (34,(57)(58)(59)(60)(61) including the acyl-CoA dehydrogenases (54,55). In particular, the polarization seen in this work shows striking parallels with that observed for the binding of cinnamoyl-CoA analogues to enoyl-CoA hydratase (34,59) and benzoyl-CoA derivatives to 4-chlorobenzoyl-CoA dehalogenase (60,61).…”

Structure and Mechanism of Citrate Synthase

“…This would favor a large positive partial charge on C-3 of oxaloacetate, making it susceptible to nucleophilic attack. Kurz and Drysdale {24,25) have reported NMR and Fourier transform IR studies which indicate that the carbonyl carbon-oxygen bond is strongly polarized.…”

Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

“…Significant binding isotope effects have also been measured in the reaction catalysed by human brain hexokinase (Lewis and Schramm, 2003), lactate dehydrogenase (LaReau et al, 1989;Gawlita et al, 1995a, b), phosphoenolpyruvate carboxylase (Gawlita et al, 1995a, b), and pyruvate kinase (Gawlita et al, 1995a, b). Furthermore, a number of spectroscopic studies have demonstrated significant polarization of substrate carbonyls upon binding enzyme and prior to catalysis that would be expected to yield corresponding isotope effects on the binding steps (Belasco and Knowles, 1980;Kurz et al, 1985;Deng et al, 1989Deng et al, , 1992Tonge and Carey, 1992). Effects such as these also can be viewed as potential contributors to any measured kinetic isotope effect intended to evaluate the transition state during the step of actual covalent rearrangement.…”