Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals

Area of Science:

• Biochemistry and structural biology
• Enzymology
• Crystallography

Background:

• Glycogen phosphorylase b catalyzes the phosphorolysis of glycogen.
• Understanding enzyme mechanisms requires visualizing reaction intermediates.
• Time-resolved crystallography offers insights into dynamic enzymatic processes.

Purpose of the Study:

• To investigate the catalytic mechanism of glycogen phosphorylase b using time-resolved X-ray diffraction.
• To capture and analyze reaction intermediates during substrate conversion.
• To compare Laue and monochromatic diffraction methods for studying enzyme kinetics.

Main Methods:

• Time-resolved Laue and monochromatic X-ray diffraction on enzyme crystals.
• Photolysis of caged 3,5-dinitrophenyl phosphate (DNPP) to initiate the reaction.
• Diode array spectrophotometry to monitor photolysis and reaction progress.
• Analysis of electron density maps to visualize substrate and product binding.

Main Results:

• Laue diffraction provided electron density maps comparable to monochromatic methods.
• Minimal catalysis observed at 3 minutes and 1 hour post-phosphate release.
• A phosphate intermediate was detected in the catalytic site.
• Product formation was slow, with mixtures of substrate and product observed under monochromatic conditions.
• A previously observed activation-related conformational change was not detected.

Conclusions:

• Time-resolved Laue crystallography is effective for studying rapid enzymatic reactions.
• The catalytic mechanism involves a distinct phosphate intermediate.
• Enzyme activation conformational changes may require specific conditions or activators.
• The caged phosphate compound may inhibit conformational flexibility.