Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.
The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)
Duke
E M
EM
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Wakatsuki
S
S
Hadfield
A
A
Johnson
L N
LN
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010763:Phosphorylase b / Q000737:chemistry* / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation; D011817:Rabbits; D013053:Spectrophotometry; D013402:Sugar Alcohols / Q000378:metabolism; D014961:X-Ray Diffraction
D009943:Organophosphorus Compounds; D010710:Phosphates; D013402:Sugar Alcohols; C074598:3,5-dinitrophenyl phosphate; C026592:2,6-anhydro-1-deoxygluco-hept-1-enitol; D010763:Phosphorylase b