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.
Wolkers
W F
WF
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Gregory
J D
JD
Churchich
J E
JE
Serpersu
E H
EH
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