title: The negative dominant effects of T340M mutation on mammalian pyruvate kinase.
abstract: A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same 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 inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. 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 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.
authors.affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1055, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Friesen
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D005635:Fructosediphosphates / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000737:chemistry*; D011817:Rabbits; D014461:Ultracentrifugation
chemical_list: D005635:Fructosediphosphates; D007527:Isoenzymes; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate