Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure

The effects of a number of cryoprotectants on the kinetic and structural pr operties of glycogen phosphorylase b have been investigated. Kinetic studie s showed that glycerol, one of the most commonly used cryoprotectants in X- ray crystallographic studies, is a competitive inhibitor with respect to su bstrate glucose-l-P with an apparent Ki value of 3.8% (v/v). Cryogenic expe riments, with the enzyme, have shown that glycerol binds at the catalytic s ite and competes with glucose analogues that bind at the catalytic site, th us preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulf oxide (DMSO) activated glycogen phosphorylase b to different extents, by st abilizing its most active conformation, while sucrose acted as a noncompeti tive inhibitor and elethylene glycol as an uncompetitve inhibitor with resp ect to glucose-l-P. A parallel experimental investigation by X-ray crystall ography showed that, at 100 K, both MPD and DMSO do not bind at the catalyt ic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for bi nding studies with glycogen phosphorylase.