Arrangement of the substrates at the active site of brain pyridoxal kinase.
The distances between enzyme-bound paramagnetic CrATP (a stable, beta, gamma-bidentate complex of Cr3+ and ATP) at the active site of sheep brain pyridoxal kinase and the protons of bound inhibitor 4-dPyr (4-deoxypyridoxine) were determined in the ternary enzyme-CrATP.4-dPyr complex by measuring the paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the protons of 4-dPyr. The correlation time for the Cr(3+)-4-dPyr dipolar interaction on the enzyme was estimated as 1.59 ns by the frequency dependence of 1/T1p of water protons. Temperature dependence of 1/T1p values indicated the fast exchange of 4-dPyr from the paramagnetic enzyme.CrATP.4-dPyr complex; hence the measured 1/T1p values can be used for metalnucleus distance determinations. The distances from the Cr3+ of the enzyme-bound CrATP to the 2-methyl (7.19 A), 4-methyl (7.18 A), and H6 proton (6.18 A) of the 4-dPyr are too great to permit a direct coordination of any group from 4-dPyr. However, these distances can be built into a model in which phosphorus of the gamma-phosphoryl group of ATP is 4 A away from the oxygen atom of the 5-CH2OH group of the 4-dPyr. This suggests that phosphorylation of pyridoxal can occur via direct transfer of the phosphoryl group between the bound substrates at the active site of pyridoxal kinase.
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
W F
WF
Wolkers
J D
JD
Gregory
J E
JE
Churchich
E H
EH
Serpersu
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D011730:Pyridoxal / Q000737:chemistry; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000648:ultrastructure*; D011736:Pyridoxine / Q000031:analogs & derivatives / Q000737:chemistry; D012756:Sheep; D013696:Temperature
D011730:Pyridoxal; C004675:4-deoxypyridoxine; D000255:Adenosine Triphosphate; D011731:Pyridoxal Kinase; D011736:Pyridoxine