An investigation of imine formation in the isocitrate dehydrogenase reaction.

The occurrence of imine formation at the active site of acetoacetate decarboxylase and several aldolases prompted an attempt to demonstrate this mechanism in the formally similar isocitrate dehydrogenase reaction. Although treatment of isocitrate dehydrogenase with NaBH4 in the presence of cr-ketoglutarate resulted in inactivation of the enzyme, reduction in the presence of labeled a-ketoglutarate did not yield labeled protein, nor was inactivation dependent on NADPH which is required for the enzymic P-H exchange of cr-ketoglutarate. A study of the isocitrate dehydrogenase reaction in HzlsO was made possible by the observation that decarboxylation of cu-ketoglutarate by HzOZ was faster than spontaneous exchange of the keto group with the medium. It was shown that succinate produced from enzymically formed a-ketoglutarate contained considerably less than 1 atom of 180. It was concluded that this tinding is not compatible with a mechanism of imine formation between keto intermediates (oxalosuccinate) and amino groups at the active site, since decarboxylation and hydrolysis of the imine to release cr-ketoglutarate would have introduced 1 atom of lsO.