Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase of Neurospora
| Autor: | Francesco M. Veronese, Emil L. Smith, Yair Degani |
|---|---|
| Rok vydání: | 1974 |
| Předmět: |
chemistry.chemical_classification
biology Stereochemistry Glutamate dehydrogenase Substrate (chemistry) Cell Biology Nicotinamide adenine dinucleotide biology.organism_classification Biochemistry Neurospora Cofactor Amino acid chemistry.chemical_compound chemistry Iodoacetamide biology.protein Thiol Molecular Biology |
| Zdroj: | Journal of Biological Chemistry. 249:7929-7935 |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/s0021-9258(19)42054-1 |
| Popis: | Selective chemical modification of sensitive amino and sulfhydryl groups was performed on purified NAD-specific glutamate dehydrogenase from Neurospora crassa (V eronese , F. M., N yc , J. F., D egani , Y., B rown , D. M., and S mith , E.L. (1974) J. Biol. Chem. 249, 7922–7928). Pyridoxal 5′-phosphate inhibited the enzyme rapidly and reversibly, resulting in the formation of 4 to 4.5 residues of e-N-(5′-phosphopyridoxyl)lysine per subunit, as determined after NaBH4 reduction. The enzyme was inactivated irreversibly by cyanate, to form a product containing 8 residues of homocitrulline per subunit. Sulfhydryl-directed reagents including p-mercuribenzoate, N-ethylmaleimide, 5,5′-dithiobis(2-nitrobenzoate), o-iodosobenzoate, and 2-nitro-5′-thiocyanobenzoate, inactivated the enzyme to a varying degree depending on the structure of the S substituent. The stoichiometry was one thiol modified per subunit by methods of activity titration, radioactive label incorporation, and spectrophotometric analysis. Tetrathionate produced an intermolecular disulfide, retaining almost full enzymic activity. No reaction was detected with either iodoacetate or iodoacetamide. Monoalkylation with N-ethylmaleimide did not alter the Vmax of the enzyme, but increased the value of Km toward both substrate and coenzyme. Consecutive modification of the sensitive amino and sulfhydryl groups indicated mutual independence of their reactivities. The chemical characteristics of the enzyme are compared with those of other glutamate dehydrogenases, including the NADP-specific enzyme from the same organism. |
| Databáze: | OpenAIRE |
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