CONFORMATIONAL CHANGES AND MECHANISM OF RESOLUTION OF GLYCOGEN PHOSPHORYLASE B

This study purports to define the role of deforming agents in promoting the release of pyridoxal 5′-phosphate from rabbit muscle phosphorylase b and the changes in structure undergone by the latter during this process. Deformation (localized reversible denaturation) of the enzyme is seen as a highly specific reaction whose course is influenced by both the nature of the cations and anions involved. While neither ammonia nor primary, secondary, or tertiary amines allowed resolution to occur, imidazole and several of its derivatives including 1-, 2-, or 4-methylimidazole were highly effective. By contrast, 4-imidazole acetic acid, histidine, was or histamine either poorly active or totally inactive. Di-, tri-, or tetracarboxylic acids which, by themselves, were quite ineffective in promoting resolution greatly accelerated this phenomenon when used in conjunction with imidazole. Specificity with respect to anions was further demonstrated when resolution was followed as a function of temperature: removal of pyridoxal 5′-phosphate increased sharply with temperature in imidazole citrate but decreased in imidazolium chloride. Data presented indicate that deforming agents cause dissociation of the enzyme into monomers and that this process unmasks the cofactor, allowing it to interact with carbonyl reagents such as lcysteine. Rapid resolution is also observed in the presence of hydroxylamine following monomerization of the enzyme with p-mercuribenzoate according to the procedure of Madsen and Cori (Madsen, N. B., and Cori, C. F. (1956), J. Biol. Chem. 223, 1075). Changes in structure upon addition of deforming agents are further evidenced by changes in ultraviolet and fluorescence spectra due to the bound cofactor; these changes are blocked by addition of effectors such as adenosine monophosphate which oppose resolution of the enzyme. No evidence was obtained that resolution proceeds through the formation of a Schiff base between the protein and the cofactor which, because of its high reactivity, could spontaneously decompose into apoenzyme and free pyridoxal 5′- phosphate. A possible scheme for the resolution of phosphorylase consistent with the data is presented. © 1969, American Chemical Society. All rights reserved.