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

Abstract

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

The Binding of Inosine Monophosphate to Escherichia coli Carbamoyl Phosphate Synthetase

“…Note that adenoylsuccinate synthetase catalyzes the production of adenoylsuccinate from IMP, aspartate, and GTP, and in this case, the nucleotide monophosphate is a true substrate. With regard to the role of IMP acting solely as an effector molecule, however, the only other observed structural example of IMP binding within an allosteric pocket is that of glycogen phosphorylase (27). In this enzyme, the ribose of the nucleotide adopts the C 3Ј -endo pucker.…”

Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure: Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase

“…In either way, binding of anion to phosphate site 1 can cause activation. The importance of anion activation is underlined by the fact that it is a widespread phenomenon among the enzymes that handle various anionic metabolites ( − ).…”

Relationships between structure, function and stability for pyridoxal 5′‐phosphate‐dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies

“…The direct comparison of pH profiles for the catalytic rates of Cc StP and the complex PL‐phosphorylase and phosphate can arguably provide mechanistic information because enzyme systems were analyzed whose active sites differed only by a minimal modification. However, any interpretation must be tempered considering that in RmGP, slightly different binding modes for cofactor‐bound and mobile phosphate groups have been detected by X‐ray crystallography [25]. The question of interest was whether differences in p K a values for covalent and noncovalent phosphate (p K a = 7.2 [23]) groups are mirrored in the corresponding pH‐rate profiles.…”