31P nuclear magnetic resonance studies of glycogen phosphorylase from rabbit skeletal muscle: ionization states of pyridoxal 5'-phosphate.

Abstract

ABSTRACT31P nuclear magnetic resonance (NMR) at 72.8 MHz has been used to study glycogen phosphorylase from rabbit muscle (1,4-a-D-glucan:orthophosphate a-glucosyltransferase, EC 2.4.1.1) at concentrations as low as 25 mg/ml, using a WH-180 wide-bore superconducting spectrometer. The use of a thio analogue for 5'-AMP and arsenate for inorganic phosphate allowed the observation of three distinct forms of enzyme-bound pyridoxal 5'-phosphate at -0.2 ppm (Form I), -2 to -3 ppm (Form II, and -3.5 ppm (Form III) relative to triethylphosphate. Conversion of I to III occurs by activation of phosphorylase either by formation-of a ternary complex of phosphorylase b with effector and arsenate or, more efficiently, by direct phosphorylation to give the a form of the enzyme. The ionization state and exposure to solvent of each of the three forms is inferred from the 31P NMR data.Pyridoxal 5'-phosphate is an essential constituent of all known a-glucan phosphorylases (1,4-a-D-glucan:orthophosphate aglucosyltransferase, EC 2.4.1.1). Reconstitution experiments with pyridoxal-P analogues (1-3) indicate the importance of the phosphate moiety in pyridoxal-P function but do not provide information on its precise role (4, 5). 31P nuclear magnetic resonance (NMR) spectroscopy gives specific information on the ionization state and the pKa values of pyridoxal-P bound to the enzyme. In addition, one can observe all other phosphorus-containing ligands involved in phosphorylase catalysis, as well as the phosphoserine group that regulates the activity of phosphorylase by interconversion of the b and a forms (6).However, the 31P NMR signals for Pi, glucose 1-phosphate, AMP, phosphoserine, and pyridoxal-P all lie in a very narrow range of ca 4 ppm. Therefore, we have substituted adenosine 5'-O-thiomonophosphate (AMP-S) for AMP and arsenate for phosphate. Adenosine 5'-O-(3-thio)triphosphate (ATP-y-S) was used to prepare phosphorylase a containing thiophosphoserine residues. Thus, it was possible to separate the signals of the various ligands and to clearly observe the pyridoxal-P resonance, which monitors the protein conformational change induced by the effector AMP or by the phosphoserine group.

Control of phosphorylase b conformation by a modified cofactor: Crystallographic studies on R‐state glycogen phosphorylase reconstituted with pyridoxal 5′‐diphosphate

“…31P-NMR studies showed that the state of ionization of the cofactor is dependent on the state of activation of the enzyme: the 5'-phosphate is a monoanion in the T-state conformation and a dianion in the R-state conformation. In the presence of substrates or inhibitors there is a change in the 31P chemical shift, which has been interpreted either as a partially protonated state or as a distorted dianion (Feldman & Hull, 1977;Helmreich & Klein, 1980;Withers et al, 1981b).…”

Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild‐type and His334→Gly mutant enzymes

“…To the extent that the shift of the 31 P resonance signal is a sensitive probe of direct contacts between the cofactor 5′‐phosphate group and bound ligands or relevant changes in active site conformation induced by ligand binding [2,38], the evidence for Cc StP suggests that the local environment of PLP phosphate remains essentially unaffected upon formation of enzyme complexes with arsenate alone and in combination with GL. By contrast, significant field shifts of the 31 P resonance signal were observed with E. coli maltodextrin phosphorylase [37], potato phosphorylase [39] and muscle glycogen phosphorylase [40] upon addition of arsenate, probably caused by electrostatic interactions between the 5′‐phosphate moiety and arsenate. The p K a for PLP phosphate in E. coli maltodextrin phosphorylase was also shifted by + 1.1 pH units upon binding of arsenate [37].…”

Glycogen phosphorylase: A multifaceted enzyme

“…Phosphorylase contains an essential cofactor, pyridoxal phosphate (4), which is bound via a Schiff base to Lys680 in the amino acid sequence (72). The 5'-phosphate group plays an obligatory role in catalysis (63) and its state of ionisation is sensitive to the state of activation of the enzyme (17). The properties of the enzyme have been the subject of several reviews (18,20,35,47,60,62).…”

“…Argl0 shifts 50 A from T state to R state reflecting the dramatic restructuring of the N-terminal peptide on phosphorylation. In T state phosphorylase b there are 11 acidic and 7 basic residues within 15/~ of the Ca atom of Serl4 (excluding residues [10][11][12][13][14][15][16][17][18][19][20]. This location of the tail provides a complimentary electrostatic environment to the basic N-terminal tail and one which is inhos- Fig.…”