Preparation and properties of a holo-lactate dehydrogenase enzyme reactor

Biochimie
E WarthC Woenckhaus

Abstract

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+...Continue Reading

References

Feb 15, 1979·FEBS Letters·P O Larsson, K Mosbach
Sep 1, 1986·Biological Chemistry Hoppe-Seyler·H G SchäferC Woenckhaus
Jun 1, 1973·Analytical Biochemistry·C Bernofsky, M Swan
Jun 1, 1959·Archives of Biochemistry and Biophysics·J M SIEGELR M BOCK

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