Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. III. Inactivation by nitration of a tyrosine residue involved in coenzyme binding.
Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme.
D000595:Amino Acid Sequence; D001665:Binding Sites; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors; D007700:Kinetics; D008024:Ligands; D008697:Methane / Q000031:analogs & derivatives*; D009243:NAD / Q000031:analogs & derivatives; D009249:NADP / Q000031:analogs & derivatives; D009491:Neurospora / Q000201:enzymology*; D010446:Peptide Fragments; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000032:analysis
D008024:Ligands; D010446:Peptide Fragments; D009243:NAD; D014443:Tyrosine; D009249:NADP; D005969:Glutamate Dehydrogenase; D013774:Tetranitromethane; D008697:Methane