Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase.
Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase [EC 1.1.1.27] was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1. The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined. Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species. At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically. Citrate activated the T. aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions. The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7. The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate. All of the modified enzymes examined were still activated by citrate similarly to the native enzyme. These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site.
D000596:Amino Acids / Q000032:analysis; D002264:Carboxylic Acids / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D002951:Citrates / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008970:Molecular Weight; D013045:Species Specificity; D013824:Thermus / Q000201:enzymology*
D000596:Amino Acids; D002264:Carboxylic Acids; D002951:Citrates; D005635:Fructosediphosphates; D006598:Hexosediphosphates; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate