Abstract
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.
Highlights
The distances between enzyme-bound paramagnetic The knowledgeof the molecularbasis of enzymatic catalysis CrATP at theactive site of sheep brain pyridoxakl inase tion on enzyme-bound substrate complexes
(7.19 4-methyl (7.18 and H6 proton(6.18 of analog, which has been succesfully used as a paramagnetic the 4-dPyrare too greato permita direct coordination probe for intersubstrate distancemeasurements [6, 7], and it of any group from 4-dPyr
This work describes an attempt to determine phosphoryl group of ATiPs 4 away from theoxygen the proximity of the bound substrates at the active site of atom of the 6-CHaOH group of the 4-dPyr. This sug- pyridoxal kinase by measuring the metal-nucleus distances gests that phosphorylation of pyridoxal can occurvia between the substratemolecules using CrATP as aparamagdirect transfer of the phosphoryl group between the netic probe. bound substratesat the active site of pyridoxalkinase
Summary
Magnetic Resonance Studies-The longitudinal relaxation rate of water protons was measured with a Seimco pulsed NMR instrument at 24.3 MHz, as described previously [14, 15], by using the 180"-T-. 90" pulse sequence of Carr and Purcell [14, 16]. The observed enhancement of the relaxation rate is defined as z = (l/Tlp)*/(l/Tlp), where 1/TIpis the paramagnetic contribution to the observed relaxation ratein the presence (*) and absence of macromolecular species. The enhancement value for the binary CrATP.4-dPyr was obtained at 169 p~ CrATP by extrapolation of the observed enhancement values a t 5-25 mM 4-dPyr concentration to infinite 4-dPyr concentration. The samples contained 50 mM MES, pH 6.0, and 75 mM
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