Lactate dehydrogenase from the extreme thermophile Thermotoga maritima.
Lactate dehydrogenase was isolated from the extreme thermophilic eubacterium Thermotoga maritima. The enzyme is stereospecific for L(+)-lactate. It represents a homotetramer of 144 kDa molecular mass, with a sedimentation coefficient of s20,w approximately 7 S. Under physiological temperature conditions, the enzyme shows high catalytic efficiency with a broad pH optimum at pH 7.0 +/- 1.0, and long-term stability up to 80 degrees C. The coenzyme, NAD+, and the effector fructose 1,6-bisphosphate [Fru(1,6)P2] increase the thermal stability: at 90 degrees C (pH 6.0), the liganded enzyme exhibits a half-life of thermal inactivation of 150 min. The enhanced rigidity of the enzyme at ambient temperature is reflected by an anomalously high stability toward guanidine denaturation: the midpoint of the equilibrium transition being 1.6 M guanidine hydrochloride. Under optimum conditions of the enzyme assay, the Michaelis constants (Km) for NADH, NAD+, pyruvate and L(+)-lactate at 55 degrees C, and in the absence of Fru(1,6)P2, are 0.03 mM, 0.09 mM, 3.7 mM and 410 mM, respectively; Fru(1,6)P2 as a positive effector shifts the Km values for pyruvate and L(+)-lactate to 0.06 mM and 25 mM, respectively. The Km values for the coenzyme are not affected. Neither Mn2+ nor other divalent cations have any activating effect. In contrast to lactate dehydrogenases from eukaryotes, the N-terminus of the enzyme from Th. maritima is not acetylated. Comparison of the 30 N-terminal amino acid residues with lactate dehydrogenase from Thermus aquaticus shows a high degree of similarity. This also holds if the two lactate dehydrogenases are compared with the glyceraldehyde-3-phosphate dehydrogenases from the same organisms.
Wrba
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Institut für Biophysik, Universität Regensburg, Federal Republic of Germany.
Jaenicke
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Huber
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Stetter
K O
KO
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013696:Temperature
D000596:Amino Acids; D006146:Guanidines; D007770:L-Lactate Dehydrogenase; D019791:Guanidine