Lactate dehydrogenase from the extreme thermophile Thermotoga maritima

Abstract

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∼ 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°C. The coenzyme, NAD+, and the effector fructose 1,6‐bisphosphate [Fru(1,6]P2] increase the thermal stability: at 90°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°C, and in the absence of Fru (1,6)P2, are 0.03 mM, 0.09 mM and 410 mM, respectively; Fru(1,6)P2, as a positive 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. Copyright © 1990, Wiley Blackwell. All rights reserved