Activity of glycogen phosphorylase in the crystalline state.
Glutaraldehyde cross-linked crystals of muscle phosphorylase a and b (alpha-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) in the tetragonal form have been shown to be catalytically active in the direction of saccharide synthesis. Precession x-ray photographs at 5.5 A resolution of a single crystal of cross-linked phosphorylase b at the hol zone indicate little change in the diffraction pattern when compared to non-cross-linked phosphorylase b under similar conditions. Non-cross-linked crystals crack and dissolve in the presence of both substrates, maltopheptaose and glucose 1-phosphate, although they are stable in the presence of each individually. These phenomena are prevented by treatment with glutaraldehyde, which causes a marked increase in mechanical stability and completely suppresses solubilization of the enzyme under our assay conditions. Diffusion of substrates into cross-linked microcrystals does not appear to be rate-limiting and assays of such crystals are linear with respect to both time and enzyme concentration. Kinetic constants for both substrates are reported. The maximal velocities of phosphorlyase a are larger than those of phosphorylase b in both the soluble and crystalline states under our assay conditions, with the above substrates. It appears that crystallization (and cross-linking) reduces maximal velocities by about 11- to 50-fold in the case of phosphorylase b and 50- to 100-fold for phosphorylase a. Little or no differences were found between the Km values for maltoheptaose or glucose 1-phosphate in the soluble or crystalline states. Kinetic data suggest that substrate binding sites are similar in both states. Although loss of catalytic efficiency points to differences in the active site of the enzyme caused by crystallization, another explanation is that the crystal is restricting a conformational change that is an essential part of the catalytic cycle.
D000818:Animals; D003460:Crystallization; D005976:Glutaral; D007700:Kinetics; D046911:Macromolecular Substances; D006005:Phosphorylases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D006005:Phosphorylases; D005976:Glutaral