Pigeon Liver Malic Enzyme

• Published in: The Journal of biological chemistry Vol. 242; no. 22; pp. 5315 - 5322
• Main Authors: Hsu, Robert Y., Lardy, Henry A., Cleland, W.W.
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25.11.1967
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99430-5

Kinetic studies with crystallized pigeon liver malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.40) gave intersecting initial velocity plots that conform to a sequential mechanism. The following product inhibition patterns were observed with TPN and malate, respectively, as variable substrates: bicarbonate, noncompetitive and noncompetitive; pyruvate, uncompetitive and uncompetitive; and TPNH, competitive and noncompetitive. These results are consistent with an ordered kinetic mechanism where TPN is added first, followed by malate, and the products are released in the order bicarbonate, pyruvate, and TPNH. Detailed analysis of the kinetic data suggest the existence of an abortive enzyme-bicarbonate complex. A reaction mechanism involving two conformational states of the enzyme is postulated. The Michaelis constants for TPN and malate, at pH 7, were found to be 1.4 µ m and 86 µ m , respectively. Apparent Michaelis constants for the back reaction were 13 m m , 6.4 m m , and 2.1 µ m for bicarbonate, pyruvate, and TPNH. Dissociation constants of TPN and TPNH were determined from kinetic studies to be 0.96 µ m and approximately 3 µ m .