Homotropic activation via the subunit interaction and allosteric symmetry revealed on analysis of hybrid enzymes of L-lactate dehydrogenase.
L-Lactate dehydrogenase from Bifidobacterium longum shows homotropic activation by pyruvate as well as heterotropic activation by fructose 1,6-bisphosphate. Hybrid enzymes were produced from the wild-type subunit and a mutant subunit, whose substrate specificity was altered to that of malate dehydrogenase, and separated to analyze the substrate-induced homotropic activation mechanism. Oxamate, a competitive inhibitor of L-lactate dehydrogenase, was used to mimic the substrate-induced activation of the wild-type subunit as "a regulatory subunit." The malate dehydrogenase activity of the mutant subunit as "the catalytic subunit" of the hybrid enzymes was measured, and the activity of the mutant subunit was activated on the addition of oxamate. Thus, we directly observed the inter-subunit homotropic activation transmitted from the wild-type to the mutant subunit. Moreover, "isomeric" hybrid enzymes that have different structural subunit arrangements but identical subunit compositions showed identical kinetic natures. This indicates that the enzyme maintains its subunit symmetry during the allosteric transition.
Fushinobu
S
S
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan.
Ohta
T
T
Matsuzawa
H
H
D001644:Bifidobacterium / Q000201:enzymology; D004789:Enzyme Activation; D007527:Isoenzymes; D007536:Isomerism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D010071:Oxaloacetates / Q000494:pharmacology; D055503:Protein Multimerization; D019289:Pyruvic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry
D007527:Isoenzymes; D010071:Oxaloacetates; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase