Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

Abstract

The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.

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).…”

Solvent-Independent Anharmonicity for Carbonyl Oscillators

“…This effect is sometimes called “bond polarization” and has been widely used to rationalize frequency shifts in condensed-phase systems. 21–23 The bond polarization effect would be expected to alter the vibrational potential and energy levels according to the Morse potential $$Vfalse(\overrightarrow{d}false)=D_{\mathrm{e}}{false[1-{\mathrm{e}}^{-\alpha false(false|\overrightarrow{d}false|-false|{\overrightarrow{d}}_{\mathrm{e}}false|false)}false]}^2$$ $${\overline{\nu }}_{01}={\omega }_0-2{\omega }_0{\chi }_{\mathrm{e}}$$ whereby χ e and/or q would be expected to change based on the solvent, thus resulting in different values of D e , ω 0 , and $false|\mathrm{normal\Delta }\overrightarrow{d}false|$, as depicted in Figure 2B. Likewise, it has been generally observed that there is a correlation between bond length and frequency shifts, 24–26 which has also been argued in support of bond polarization.…”

FTIR studies of hydrogen bonding between α,β-unsaturated esters and alcohols

“…The [3,4], citrate synthase [5], phospholipase A2 [6], chloramphenicol acetyltransferase [7], lactate dehydrogenase [8], and ketosteroid isomerase [9]. In addition, several groups have used substrates based on o, P-unsaturated arylacryloyl esters to probe the active sites of serine and cysteine proteases [l The model studies are performed using a compound that resembles the enzyme-substrate complex as closely as possible.…”