Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase of Neurospora

A specific cysteine residue in NAD-dependent glutamate dehydrogenase from Neurospora crassa was labeled with N-ethyl[1-14C]maleimide. Inactivation of the modified enzyme with pyridoxal 5'-phosphate followed by reduction with NaBH4 resulted in the preferential labeling of 4 to 4.5 lysine residues per subunit. Tryptic hydrolysis of the doubly labeled enzyme yielded a single radioactive peptide containing a S-(N-ethyl[14C]succinimido)cysteine residue, and its sequence was identified as Val-Leu-Cys*-Asp-Asp-Thr-Asn-Val-Thr-Leu-Pro-Asp-Gly-(Val,Gln,Asn)-His-Gly-Thr-Ala-Phe-Arg. Five fluorescent peptides containing e-N-(5'-phosphopyridoxyl)lysine residues were isolated from the digest, and their sequences were determined as: (a) Ser-Lys*-Glu-Ala-Tyr-Gln-Ile-Asn-Ala-Arg; (b) Ala-Thr-Lys*-Asn-Thr-Lys; (c) Leu-Glu-Lys*-Lys; (d) Leu-Ala-Lys*-Ala-Arg; (e) Lys*-Asn-Lys. None of the sequences of the labeled peptides showed homology with regions containing essential lysine residues or the cysteine residues in other glutamate dehydrogenases previously described; however, there may have been additional labeled peptides representing different sequences that were not obtained.