Chemical modification of glutamate dehydrogenase by 2,4,6-trinitrobenzenesulphonic acid.
1. Modification with 2,4,6-trinitrobenzenesulphonic acid was studied for its effect on the structure, activity and response to regulatory effectors of ox liver glutamate dehydrogenase. 2. The modification affected amino groups only, and the relative reactivities of the amino groups of the enzyme are described. 3. A biphasic inactivation of the enzyme was observed and analysis of the course of inactivation and of modification showed that the rapid reaction of one amino group/subunit leads to loss of 80% of the enzymic activity. 4. NADH retarded the inactivation by 2,4,6-trinitrobenzenesulphonic acid, the protection increasing with NADH concentration. This, together with the previous observation, suggests that the rapidly reacting group is essential for the activity of the enzyme. 5. The effects of modification on the optical-rotatory-dispersion and sedimentation behaviour of the enzyme were studied. 6. The enzyme's response to the allosteric effector GTP was rapidly lost on modification, whereas its response to ADP was unaffected. Comparison of the inactivation and desensitization suggests that the reactive amino group is essential for both activity and GTP response, and that only a completely unmodified enzyme oligomer responds fully to GTP. 7. The merits of chemical-modification studies of large enzymes are discussed critically in connexion with the interpretation of these results.
D000227:Adenine Nucleotides; D000588:Amines; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006150:Guanine Nucleotides; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD; D009578:Nitrobenzenes / Q000494:pharmacology*; D009905:Optical Rotatory Dispersion; D013451:Sulfonic Acids / Q000494:pharmacology*; D014461:Ultracentrifugation
D000227:Adenine Nucleotides; D000588:Amines; D006150:Guanine Nucleotides; D009578:Nitrobenzenes; D013451:Sulfonic Acids; D009243:NAD; D005969:Glutamate Dehydrogenase