We propose to determine the crystal structure of the R (active) conformation of glycogen phosphorylase at high resolution, and elucidate the structures of an enzyme-substrate complex analog and a covalent enzyme intermediate. We expect to carry out a detailed comparison of this structures with that already determined for the inactive T state and its effector complexes. Phosphorylase is a complex (97.4 KDalton) allosteric enzyme regulated by a number of physiologically important ligands and by phosphorlyation/dephosphorylation under neural and hormonal control. Our goal is to understand the nature of the T yield R transition in this enzyme. We hope to elucidate the mechanism of allosteric regulation by effectors and covalent introconversion as well as the mechanism by which conformation changes are communicated among effector sites within and between subunits. This research is also intended to provide a structural basis on which models of catalysis can be evaluated. The structure to be determined is that of Pyridoxalpyrophosphoryl phosphorylase b (PLPPb), a putative analog of the binary complex between phosphorylase and one of its substrates, orthophosphate (Pi). Withers and Madsen (4) have shown this enzyme to be locked into an R-state conformation in the presence of AMP. Crystals (space group P2-1,2-1,2,a a=169.9, b=209.8, c= 123.3) were obtained of PLPPb in the presence of AMP and the second substrate maltopentaose. PLPPb diffract to at least 2.4 angstroms resolution and a complete 3.0 angstroms data set for the native enzyme, and 3.5 angstroms sets for two isomorphous heavy atom derivatives have been measured using the Multiwire area detector in the laboratory of N-H. Xuong at U.C. San Diego. We propose, a) to determine the native structure at 3.0 angstroms then extend the analysis to high, near-atomic resolution; b) Measure a data set for the phosphorylated PLPPa enzyme to at least 3.0 anstroms, so to elucidate the structural role of phosphorylation in allosteric regulation (this structure is to be compared with that of PLPPb, T-state Phosphorylase a (7) and with T-state phosphorylase b determined in Jouise Johnson's laboratory (3); and c) to determine the structures to 3.0 angstroms resolution of the enzyme-phosphoglucoside substrate analog PLPP-Glucoside phosphorylase b and, if possible, stabilized catalytic intermediate, Glucosyl PLPP phosphorylase.
