title: Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.
abstract: N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.
authors.affiliation: a Cell and Molecular Biology program ; Michigan State University ; East Lansing , MI USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Beauchamp
authors.forename: Claire
authors.initials: C
authors.lastname: Vieille
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits; D012685:Sepharose / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism
chemical_list: D009243:NAD; D012685:Sepharose; D010088:Oxidoreductases; D013401:Sugar Alcohol Dehydrogenases; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase; C020672:glycerol dehydrogenase; D005969:Glutamate Dehydrogenase
keywords: DHA, dihydroxyacetone; LmG6PDH, Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase; N6-CM-NAD, N6-carboxymethyl-NAD; NAD; NAD, nicotinamide adenine dinucleotide; TmGlyDH, Thermotoga maritima glycerol dehydrogenase; TmMtDH, Thermotoga maritima mannitol dehydrogenase; analog; bGDH, bovine liver type III L-glutamic dehydrogenase; catalysis; cofactor; dehydrogenase; immobilize; rLDH, rabbit muscle type XI L-lactate dehydrogenase; sepharose; yADH, Saccharomyces cerevisiae alcohol dehydrogenase