Observations on the Function of Pyridoxal-5-Phosphate in Phosphorylase

Illingworth, Barbara; Jansz, H.S.; Brown, David H.; Cori, Carl F.

doi:10.1073/pnas.44.12.1180

Cited by 65 publications

(20 citation statements)

References 8 publications

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“…Reconstitution experiments with pyridoxal-P analogues (1)(2)(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.…”

mentioning

confidence: 99%

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

Feldmann¹,

Hull²

1977

Proc. Natl. Acad. Sci. U.S.A.

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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.

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confidence: 99%

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

Feldmann¹,

Hull²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…However, even when the enzyme concentration is very high (5 mg per ml), there is still an absolute lag period during which no liberation of Pi can be detected with a method sensitive to a change of 0.04 ,umole Pi per ml of reaction mixture. In a particular experiment Pi was found to the extent of about 1 C,,,6 tig/ml glycogen; D, 14 j~g/ml glycogen. In Fig.…”

Section: '~54 Admentioning

confidence: 80%

“…=± [G]n + I + Pinorganic (1) [G]. stands for a "primer" which can accept glucose residues from G-1-P in the elongation of chains.…”

Section: '~54 Admentioning

confidence: 99%

“…Comparison of chemically synthesized G-1-P (0O) and G-1-P prepared from starch (0). Phosphorylase a eluted from DEAE cellulose column and recrystallized from EDTA-mercaptoethanolglycerophosphate, 1.06 mg/ml; 0.04 M G-1-P; 0.001 M 5 -AMP; LU act. 3,000 units/mg) was incubated at 30c with G-1-P and 5'-AMP at pH 6.7.…”

Section: '~54 Admentioning

confidence: 99%

“…An iodine test at that time showed that no chromogenic polysaccharide chains remained. The digests were heated in boiling water and deionized by passage through 1 the columns with water, the eluates were evaporated to dryness, taken up in 1.5 ml of water, and one-fifth of the total solution of each (corresponding to 5.3 and 8.2 mg polysaccharide glucose) was spotted on Whatman No. 1 paper for descending chromatography.…”

Section: '~54 Admentioning

confidence: 99%

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