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Studies of glutamate dehydrogenase. Methionine-169: the preferentially carboxymethylated residue
Abstract:
In phosphate buffer at pH 7.0, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide or iodoacetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivities the enzyme irreversibility by alkylation. Combined addition of the coenzyme NADH and the substrate 2-oxoglutarate or the effector GTP protects against this inactivation. The alkylation reaction is independent of pH between pH 6-9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo-[2-14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine-169 and only 7% into cysteine-115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.
Insights
Beef liver glutamate dehydrogenase activity is irreversibly inhibited by iodoacetate alkylation. This inactivation is prevented by coenzyme NADH, substrate 2-oxoglutarate, or effector GTP, suggesting a protective mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Beef liver glutamate dehydrogenase is a key enzyme in amino acid metabolism.
- Understanding enzyme inactivation mechanisms is crucial for biochemical research and drug development.
Purpose of the Study:
- To investigate the inactivation mechanism of beef liver glutamate dehydrogenase by alkylating agents.
- To identify the specific residues involved in the inactivation process.
- To explore protective strategies against enzyme inactivation.
Main Methods:
- Enzyme activity assays using various reagents like iodoacetate, N-ethylmaleimide, and 5,5'-dithio-bis(2-nitrobenzoic acid).
- pH-dependent inactivation studies.
- Radioactive labeling with iodo-[2-14C]acetic acid followed by amino acid analysis.
- Denaturation and thiol group titration.
Main Results:
- Iodoacetate irreversibly inactivates beef liver glutamate dehydrogenase via alkylation.
- The coenzyme NADH, substrate 2-oxoglutarate, and effector GTP protect the enzyme from inactivation.
- Alkylation occurs independently of pH between 6-9, suggesting non-amino, imidazole, or phenolic groups are involved.
- Titration indicates loss of approximately one thiol group per polypeptide chain upon inactivation.
- Radioactive labeling reveals that both methionine (50% at residue 169) and cysteine (7% at residue 115) are alkylated, with minor labeling of other residues.
Conclusions:
- Iodoacetate inactivation of beef liver glutamate dehydrogenase involves alkylation of both methionine and cysteine residues.
- The protective effect of NADH, 2-oxoglutarate, and GTP highlights the enzyme's conformational changes during catalysis.
- The study discusses potential reasons for erroneous thiol group titration results in similar experiments.