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 p hosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started follow ing liberation of phosphate from a caged phosphate compound, 3,5-dinit rophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flash es 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 a t 3 min from the start of reaction, and data collection comprised thre e 800-ms exposures. Careful data processing of Laue photographs for th e large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference map s obtained from the Laue measurements showed that very little catalysi s had occurred 3 min and 1 h after release of phosphate, and a distinc t peak consistent with the position expected for phosphate, in the att acking 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 phosphat e in the crystal from 5 flashes with a xenon flashlamp within 1 min fo r the reaction to go to completion within the time scale of the monoch romatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously wi th 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 phosphorylas e and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conform ational change, and because activators of the enzymic reaction were no t present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer p hotons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through cryst allographic 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, con sistent with approximate calculations for the likely rate of the react ion in the crystal. In these experiments in which only partial reactio n had occurred, there was connecting density between the phosphate and the O2 hydroxyl of the sugar substrate. The recent evidence for the i nfluence of the O2 hydroxyl on the catalytic mechanism is discussed.