Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Chang, Yen Chung; McCalmont, Timothy H.; Graves, Donald J.

doi:10.1021/bi00290a017

Cited by 30 publications

(38 citation statements)

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“…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).…”

Section: Kineticssupporting

confidence: 91%

“…Initial rates were measured in the presence of varying concentrations of phosphite and Glc-1-P at constant saturating concentrations of AMP (1 mM) and glycogen (1 %) and data analyzed by double-reciprocal plots (data not shown). Straight lines were obtained approximately converging at the x axis, in agreement with the proposed bi-reactant sequential mechanism (Chang et al, 1983). The equation based on this kinetic model (Scheme I), similar to one described previously by Segel (1975) was added to GraFit (Leatherbarrow, 1992) using the built-in equation editor.…”

Section: Kineticssupporting

confidence: 76%

“…The pH dependencies of the PL-GP using phosphite (pK, = 6.6) or fluorophosphate as activator anions are also unaltered, despite the difference in the pK, values of these anions . 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.…”

mentioning

confidence: 99%

“…The enzyme reconstituted with pyridoxal (PL) is inactive, but addition of dianions such as phosphite, fluorophosphate, phosphate, and some analogues can confer activity, indicating that these anions may bind at the subsite occupied by the coenzyme 5"phosphate in the native GP (Pamsh et al, 1977;Chang et al, 1983). The observation that phosphite activation of PL-GP can be reversed by pyrophosphate, a potent inhibitor of the enzyme, strictly competitive with both anion and Glc-I-P (Pamsh et al, 1977;Chang et al, 1983), implied the adjacent positions of activator anion and substrate phosphate in the catalytic site. 31P-NMR studies have shown that the 5"phosphate group of PLP is a monoanion in the T-state conformation and a dianion in the R-state conformation.…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

et al. 1996

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It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5"phosphate (Chang YC, McCalmont T, Graves DJ. 1983. Biochemistry 224987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase 6, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P43212, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 8, resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5"phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.Keywords: binding, fluorophosphate, phosphate, phosphite, pyridoxal phosphorylase, T state Glycogen phosphorylase (GP) catalyzes the first step in the intracellular degradation of glycogen into glucose 1 -phosphate (Glc-1 +).It is controlled by shifts in an equilibrium between several conformational states, depending on the phosphorylation state and the binding of various allosteric effectors, and ranging from a low affinity T state to a high affinity R state (in the nomenclature of

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Section: Kineticssupporting

confidence: 91%

Section: Kineticssupporting

confidence: 76%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 1996

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A novel coenzymatic function of pyridoxal 5′‐phosphate

Fukui

Tagaya

1986

BioEssays

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Pyridoxal 5′‐phosphate, the vitamin B6 derivative, acts as the coenzyme of many enzymes involved in amino acid metabolism. Exceptionally, this compound was found covalently bound to glycogen phosphorylase, the key enzyme in the regulation of glycogen metabolism. Although it is essential for the function of phosphorylase, its direct role has remained an enigma. We have recently found that the glucose moiety of pyridoxal (5′)diphospho (1)‐α‐D‐glucose, a conjugate of pyridoxal 5′‐phosphate and glucose 1‐phosphate through a pyrophosphate linkage, is transferred to the nonreducing end of glycogen, forming a new α‐1,4‐glucosidic linkage. This finding emphasizes the importance of the direct phosphate‐phosphate interaction between the coenzyme and the substrate in the phosphorylase catalytic reaction. We have proposed a catalytic mechanism for phosphorylase in which the phosphate group of pyridoxal 5′‐phosphate acts as an electrophile to the phosphate group of glucose 1‐phosphate. This appears to represent the first instance of the direct involvement of a phosphate group in catalysis by enzymes.

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