An essential arginyl residue in yeast hexokinase

Area of Science:

• Biochemistry
• Enzymology
• Protein chemistry

Background:

• Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) plays a crucial role in glucose metabolism.
• Understanding enzyme inactivation mechanisms is vital for biochemical research and drug development.

Purpose of the Study:

• To elucidate the mechanism of yeast hexokinase A inactivation by phenylglyoxal.
• To identify the specific amino acid residue modified and its role in enzyme activity.

Main Methods:

• Kinetic analysis of enzyme inactivation under varying phenylglyoxal concentrations.
• Radiolabeling studies using [14C]phenylglyoxal to quantify reagent incorporation.
• Assessment of enzyme conformational changes using biophysical techniques.
• Substrate protection assays to determine the role of active site residues.

Main Results:

• Phenylglyoxal inactivation follows pseudo first-order kinetics, suggesting a reversible enzyme-reagent complex.
• Enzyme activity loss correlates with the incorporation of one phenylglyoxal molecule per 50,000-dalton subunit.
• No significant conformational changes were observed during inactivation.
• Inactivation is attributed to the modification of a specific arginyl residue.

Conclusions:

• Phenylglyoxal selectively modifies an arginyl residue in yeast hexokinase A, leading to inactivation.
• The modified arginyl residue is crucial for enzyme function and likely interacts with the terminal phosphoryl group of ATP or glucose 6-phosphate.
• These findings provide insights into the active site structure and catalytic mechanism of yeast hexokinase A.