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The spontaneous hydrogen-deuterium exchange of the methylene group of malonyl-thioesters was investigated by nuclear-magnetic-resonance (NMR) spectroscopy using the model compound S-malonyl-N-acetylcysteamine. The half life of the methylene proteins is 12 to 16 min in 0.1 M K-phosphate buffer at pH 6.5 to 7.0 at 25 degrees C, the conditions of maximal activity of fatty acid synthetase from yeast. Proton catalysis was used for the quick preparation of deuterium- and tritium-labeled malonylthioesters. Compared with malonyl-CoA, dideutero-malonyl-CoA had no primary isotope effect on the reaction velocity of the yeast enzyme catalysed fatty acid synthesis, in which the rate limiting step is the condensation reaction. Although deuterium oxide had a solvent isotope effect, there was no difference in reaction velocities between malonyl CoA and dideuteromalonyl CoA in deuterium oxide. The condensation reaction was investiaged separately from the overall fatty acid synthesis using beta-ketoacyl-acyl-carrier-protein (ACP) synthetase (condensing enzyme) of Escherichia coli. The condensation reaction with deuteromalonyl-ACP had no kinetic isotope effect, in agreement with the observations on the overall reaction. However, in this case no solvent isotope effect was observed with 2H2O. When the condensation reaction was carried out in the presence of tritiated water, there was no incorporation of label into the reaction product acetoacetyl-thioester, excluding proton exchange with the solvent. The results exclude a mechanism for the condensation reaction involving a malonyl carbanion and its acylation as intermediates in the sense of an organic-chemical malonic ester synthesis, and they indicate that the condensation reaction follows a concerted mechanism: The formation of the new carbon-carbon bond is coupled with the cleavage of the carboxyl bond of the malonyl group.
Arnstadt et al. (Tue,) studied this question.