Kinetic evaluation of substrate-dependent origin of the lag phase in soybean lipoxygenase-1 catalyzed reactions.
We have measured, under identical conditions, the time courses for the native lipoxygenase (Fe2+ form)-catalyzed conversion of linoleic acid into 13-hydroperoxy-9,11-octadecadienoic acid (HPOD) and the oxidation of the Fe2+ form of enzyme to the Fe3+ form (in 0.1 M borate buffer, pH 10.0, at 25 degrees C) using a stopped-flow spectrophoto/fluorometer. The experimental results clearly demonstrate that the time course for the appearance of the reaction product is much shorter than that for the conversion of E-Fe2+ to E-Fe3+; the latter process involves a pronounced lag phase whereas the former does not. This suggests that the Fe2+ form of the enzyme is also catalytically active and that the origin of the lag phase is not intrinsic to the oxidation of the enzyme bound iron cofactor. When the Fe3+ form of the enzyme is utilized to investigate the time course of product formation, the lag phase was observed at substrate concentrations higher than 20 microM. The magnitude of this lag phase increases with the increases with the increase in the initial concentration of the substrate (at least up to the range where substrate is not dimerized) and decreases in the presence of increasing concentrations of HPOD (exogenously added to the reaction mixture). No lag phase is evident at substrate concentrations in the range of 10 microM or less. We have examined the effects of varied concentrations of substrate and product on the initial rates of the lipoxygenase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
Z X
ZX
Biochemistry Department, North Dakota State University, Fargo 58105.
Killilea
S D
SD
Srivastava
D K
DK
D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013997:Time Factors
D007527:Isoenzymes; D008084:Lipoxygenase