Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies.
Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8'-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart L-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS.
Forde
Jessica
J
Department of Applied Biology and Chemistry, Institute of Technology, Kilkenny Road, Carlow, Ireland.
Oakey
Laura
L
Jennings
Linda
L
Mulcahy
Patricia
P
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D050803:Leucine Dehydrogenase; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry*
D009243:NAD; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D005969:Glutamate Dehydrogenase; C073215:phenylalanine dehydrogenase (NAD); C061421:glutamate dehydrogenase (NAD(P)+); D050803:Leucine Dehydrogenase