J Biol Chem, Vol. 274, Issue 7, 3941-3945, February 12, 1999

Mitochondrial Citrate Synthase Is Immobilized in Vivo

From the University of Cambridge, Department of Biochemistry, Old Addenbrooke's Site, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom

The enzymes of the tricarboxylic acid cycle in the mitochondrial matrix are proposed to form a multienzyme complex, in which there is channeling of substrates between enzyme active sites. However no direct evidence has been obtained in vivo for the involvement of these enzymes in such a complex. We have labeled the tricarboxylic acid cycle enzyme, citrate synthase 1, in the yeast Saccharomyces cerevisiae, by biosynthetic incorporation of 5-fluorotryptophan. Comparison of the ¹⁹F NMR resonance intensities from the labeled enzyme in the intact cell and in cell-free lysates indicated that the enzyme is motionally restricted in vivo, consistent with its participation in a multienzyme complex.