Abstract
The role of pyridoxal 5'-phosphate in the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1) remains unresolved despite extensive investigation. A previous report from this laboratory (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3716-3719) provided evidence for the direct interaction between the two phosphate groups of the coenzyme and a substrate alpha-D-glucose 1-phosphate. When apophosphorylase is reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose, the enzyme can transfer the glucose moiety to glycogen just as in the normal catalysis. We have studied the kinetics of the glucosyltransfer from this compound to glycogen. The normal and mimic reactions were similar in their kinetic parameters for glycogen and AMP and their activation energies. AMP and other nucleotides that activate the normal reaction could activate the mimic reaction as well. The log k/pH plot for the mimic reaction gave a bell-shaped curve with pKa = 6.90 and pKb = 8.84 at 25 degrees C. The apparent heats of ionization of the corresponding groups having the pKa and pKb were 6.9 and 3.0 kcal/mol, respectively. Reversibility of the glucosyltransfer from the coenzyme-substrate conjugate to glycogen could not be demonstrated, possibly because the equilibrium of this reaction lies further to the polysaccharide synthesis. Based on these and other data which confirm the role of the phosphate group of the coenzyme as an electrophile, we discuss the catalytic mechanism of glycogen phosphorylase. It is suggested that the imidazoyl group of His-376 acts as a nucleophile attacking the anomeric carbon of the substrate glucose 1-phosphate.
Highlights
We have studied the kineticsof the glucosyltransfer [6],and that thecoenzyme phosphate is the functional group from this compound to glycogen
We have recently studied the catalytic mechanism of phos
Based on these and other datawhich confirm the role of the phosphate group of the coenzyme as an electrophile, we discuss the catalytic mechanism of glycogen phosphoryIase
Summary
Effect of Glycogen-The glucosyltransfer from PLPP-a-Glc to glycogen, in the absence of AMP, by rabbit muscle phosphorylase b obeyed the first order kinetics at any glycogen concentration tested (Fig. lA).From the Hill plot (Fig. lB), K, (K, refers to the substrate concentration giving k = k / 2 throughout this paper) and the Hill coefficient for glycogen were evaluated to be 0.52 mM and 0.91, respectively. Hill plot (Fig. 28), K,,a,nd theHill coefficient for AMP were calculated to be 8.0 p~ and 1.4, respectively These values are similar to those of the normal phosphorylase b reaction determined under similar reaction conditions at the saturated concentration of Glc-1-P, 22 ptd and 1.4, respectively. It should be noted that the two pK values obtained must be derived from amino acid side chains in the enzyme protein, since the pyridoxal-proximal phosphate group of PLPP-a-Glc stays monoanionic in the pH range applied presently and can neither accept nor donate a proton
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