The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.
Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D001120:Arginine / Q000502:physiology*; D002074:Butanones / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D011485:Protein Binding
D002074:Butanones; D009243:NAD; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D001120:Arginine; D005969:Glutamate Dehydrogenase