Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.
Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D001409:Bacillus cereus / Q000201:enzymology*; D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD; D011487:Protein Conformation; D011489:Protein Denaturation; D012685:Sepharose; D014227:Triazines
D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D014227:Triazines; D009243:NAD; C012842:Cibacron Blue F 3GA; D012685:Sepharose; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase