Covalently activated glycogen phosphorylase: a phosphorus-31 nuclear magnetic resonance and ultracentrifugation analysis.

Glycogen phosphorylase b reconstituted with pyridoxal pyrophosphate in place of the natural coenzyme, pyridoxal phosphate, is shown to exist in a more activated (R) conformation than does native phosphorylase. Addition of nucleotide activator to the reconstituted enzyme traps it totally in this activated conformation. These conclusions were arrived at on the basis of tertiary structural information obtained from 31P nuclear magnetic resonance studies, which allowed measurement of the nucleotide binding constant, and on the basis of quaternary structural information obtained via ultracentrifugal analysis of the enzyme in the presence of various effectors. Control experiments were performed with another modified form of the enzyme, pyridoxal phosphorylase. It is suggested that the transition-state analogue pyridoxal pyrophosphate, bound at the active site, mimics the actual configuration of enzyme plus substrate achieved during the normal catalytic reaction and therefore traps the enzyme in an activated conformation. These findings agree well with recent results obtained with the alternate transition-state analogue pyridoxal pyrophosphate glucose [Withers, S. G., Madsen, N. B., Sykes, B. D., Takagi, M., Shimomura, S., & Fukui, T. (1981) J. Biol. Chem. 256, 10759] and therefore provide further evidence for the "interacting phosphates" hypothesis presented in the latter paper.