Preparation and properties of a holo-lactate dehydrogenase enzyme reactor.
NAD+ was the base material for syntheses of coenzyme analogs with reactive groups bound to N6 of the adenine moiety via spacers that are 3-17 A long. These analogs were used for the modification of dehydrogenases. Aromatic imidoesters and acyl azides are suitable reactive groups, which form covalent amidinium or amide bonds with amino acid residues such as the epsilon-amino groups of lysines. The catalytic function of the modified protein decreased only slightly. Coenzymes that are linked via a spacer to carboxyl and amino groups are fixed to the protein by means of carbodiimides and hydroxysuccinimide. Coenzyme-bound aromatic imidoesters with spacer lengths of more than 12 A were incorporated to the extent of 60% at the active site. Aliphatic imidoesters proved to be inefficient for protein modification because of fast hydrolysis. Fixing of coenzyme analogs containing appended carboxyl or amino groups to enzyme in the presence of carbodiimides resulted in a decrease of enzyme activity. Modified lactate dehydrogenase and L-alanine dehydrogenase formed an enzyme reactor for the production of L-alanine in the absence of free NAD+. Both enzymes were cross-linked by dimethyl suberimidate in the presence or absence of NAD+, bis-NAD+, pyruvate, and oxamate. Site-to-site directed cross-linking yielded a reaction mixture from which four protein fractions were obtained by isoelectric focusing; one of these showed a cycling rate of 600 h-1.
Warth
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Klinikum der Johann Wolfgang Goethe-Universität, Gustav Embden-Zentrum der Biologischen Chemie, Frankfurt, FRG.
Jacobi
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Woenckhaus
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D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases; D055598:Chemical Phenomena; D002621:Chemistry; D003067:Coenzymes; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D007096:Imidoesters; D007525:Isoelectric Focusing; D007770:L-Lactate Dehydrogenase / Q000138:chemical synthesis*; D015394:Molecular Structure; D009243:NAD; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity
D003067:Coenzymes; D003432:Cross-Linking Reagents; D006834:Hydrazines; D007096:Imidoesters; D009243:NAD; D007770:L-Lactate Dehydrogenase; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase