Structure and Function of L-Lactate Dehydrogenases from Thermophilic and Mesophilic Bacteria. I) Isolation and Characterization of Lactate Dehydrogenases from Thermophilic and Mesophilic Bacilli

Abstract

Lactate dehydrogenase from thermophilic bacilli (Bacillus stearothermophilus, B. caldotenax) and from mesophilic bacilli (Bacillus X1, B. subtilis) were isolated by a 2-step purification procedure. Only 1 type (LDH-P4), composed of 4 identical subunits (MW 34,000 or 36,000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N[amino]-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.