THE NEGATIVE DOMINANT EFFECTS OF T340M MUTATION ON MAMMALIAN PYRUVATE-KINASE

A fundamental issue in allosteric regulatory enzymes is the identifica tion of pathways of signal transmission. Rabbit muscle and kidney pyru vate kinase isozymes are ideal to address this issue because these iso zymes exhibit different enzymatic regulatory patterns, and the sequenc e differences between these isozymes have identified the amino acid re sidues that alter their kinetic behavior. In an earlier study, Cheng e t al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Ch em. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the sam e mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or in hibitor. Because the diversity in the primary sequence between these t wo isozymes does molt alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative ef fect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subuni t interface. Thus, this study has identified a communication network a mong the active site, residue 340, and the 1,2 subunit interface.