Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

Abstract

A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-19F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDPL showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL [Chang, Y. C., & Graves, D. J. (1985) J. Biol. Chem. 260, 2709-2714], except the Km of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower Vmax value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986) Biochemistry 25, 1932-1939].(ABSTRACT TRUNCATED AT 250 WORDS)

Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal

“…The program then determines all the dissociation constants and V,,,, simultaneously by fitting all of the data at once. The values were similar to those reported previously (Parrish et al, 1977;Chang et al, 1983Chang et al, , 1987 except for those for Glc-I-P. The K,y and a K , values were reported to be 18 and 22 mM, respectively (Chang et al, 1983).…”

“…Furthermore, "F-NMR spectra of the bound fluorophosphate activator also showed that no change in its ionization state occurred on binding or during catalysis (Chang et al, 1983), a result not in accord with a role for the cofactor 5"phosphate as a proton donor in catalysis. Based on these results and other results from kinetic and 19F-NMR experiments of GPb reconstituted with 6-fluorinzted PL and DPL, Chang et al (1987) suggested that the cofactor 5'-phosphate is mainly a structural determinant holding other groups in the correct orientation for catalysis. PL-GPb provides a good enzyme derivative to study possible functions of the cofactor 5"phosphate in GP catalysis (Chang et al, 1983(Chang et al, , 1987.…”

“…Based on these results and other results from kinetic and 19F-NMR experiments of GPb reconstituted with 6-fluorinzted PL and DPL, Chang et al (1987) suggested that the cofactor 5'-phosphate is mainly a structural determinant holding other groups in the correct orientation for catalysis. PL-GPb provides a good enzyme derivative to study possible functions of the cofactor 5"phosphate in GP catalysis (Chang et al, 1983(Chang et al, , 1987. On the basis of tertiary and quaternary structural information deduced from 31P-NMR and ultracentrifugation data (Withers et al, 1982b), it has been shown that PL-GPb is an allosteric enzyme whose R state can quite easily be pushed into a T-state conformation by phosphite and Glc.…”

“…Thus, the dissociation constant of phosphite is approximately ten times higher, and V,, decreases from 15.4 (PL-GPb) to 2.8 units/mg in the DPL-GPb (Chang et al, 1987). These differences were attributed to the lack of a hydrogen bond between the 0-5 of PL and phosphite in the latter derivative (Chang et al, 1983(Chang et al, , 1987. The crystallographic results on the PL-GPb-anion-Glc-IMP complexes indicate that the closest distance between 0-5 and activating phosphate oxygen OP-3 (Table 3) is 2.7 A, which is sufficiently short to assume a hydrogen bond.…”

“…Substitution of this group in PL-GPb by a -H in DPL-GPb affects both phosphite binding and enzymic activity. Thus, the dissociation constant of phosphite is approximately ten times higher, and V,, decreases from 15.4 (PL-GPb) to 2.8 units/mg in the DPL-GPb (Chang et al, 1987). These differences were attributed to the lack of a hydrogen bond between the 0-5 of PL and phosphite in the latter derivative (Chang et al, 1983(Chang et al, , 1987.…”