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Abstract
The l-leucine dehydrogenase (LeuDH, EC 1.4.1.9) from the extremely thermophilic Bacillus caldolyticus (DSM 405) has been purified to homogeneity and crystallized. Its physical and biochemical properties, such as subunit structure, M r, amino acid composition, isoelectric point, heat stability, pH-optima, K m values for coenzymes and substrates, and pattern of inhibition have been determined. The native enzyme is an octamer (M r 320000) composed of identical subunits (M r 39000) which contain one intrachain disulphide bridge. Only aliphatic amino acids were accepted as substrates and a preference was exhibited for branched-chain residues. Inhibition occurred only upon incubation with thiol reagents such as HgCl2 and p-mercuribenzoate, and pyridoxal 5'-phosphate. The LeuDH was very thermostable, with 50% residual activity remaining after 30 min incubation at 80 C. Its properties, as well as amino acid composition and N-terminal sequence, were compared with those of the phylogenetically related LeuDH from the mesophile Bacillus cereus. Both enzymes displayed very similar properties: the quaternary structures were identical, amino acid composition alike, the N-terminal sequence showed 62% homology for the first 19 residues, and K m values for the different substrates differed by a factor of two or less, indicating that the active centres had a similar structure. Thus, the only major difference observed was the much higher stability of the thermophilic enzyme to denaturation by heat and organic solvents.
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