The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

Area of Science:

• Biochemistry
• Enzymology

Background:

• Glutamate dehydrogenase is a key enzyme in amino acid metabolism.
• Understanding its regulation is crucial for metabolic studies.

Purpose of the Study:

• To investigate the effect of diethyl pyrocarbonate modification on glutamate dehydrogenase activity.
• To elucidate the mechanism of enzyme activation and its regulatory properties.

Main Methods:

• Chemical modification of glutamate dehydrogenase with diethyl pyrocarbonate.
• Enzyme kinetics assays to determine specific activity and binding constants.
• Investigation of the effects of other ligands (NADH, GTP, NAD+, ADP) on the modified enzyme.

Main Results:

• Diethyl pyrocarbonate reacts with glutamate dehydrogenase in a 1:1 molar ratio, forming N(1)-carbethoxyhistidine.
• The modification results in a 1.4-2.0-fold increase in specific activity, which is prevented by NADH.
• NADH binding and enzyme molecular weight remain unchanged, but the activated enzyme shows a fivefold decrease in K(s) for ADP, indicating further allosteric activation.
• The activation is not consistent with NADH dissociation being rate-limiting.

Conclusions:

• Diethyl pyrocarbonate activates glutamate dehydrogenase through a mechanism that does not involve NADH dissociation.
• The modification alters the allosteric regulation of the enzyme, particularly enhancing ADP activation.
• The study provides insights into the complex regulatory mechanisms of glutamate dehydrogenase.