|
A series of kinetic isotope effect experiments were performed with the goal of
understanding the nature of rate-limiting steps in the soybean lipoxygenase-1
(SBL-1) reaction. SBL-1 was reacted with linoleic acid (LA) and deuterated
linoleic acid (D-LA) under a variety of experimental conditions involving
changes in temperature, pH, viscosity, and replacement of H2O with D2O. The
extrapolated intrinsic primary H/D isotope effect can be estimated to be
possibly as large as 80. This value is probably the largest isotope effect
published for an enzymatic reaction, and much larger than that predicted from
semiclassical models. Due to this large primary isotope effect, the C-D bond
cleavage fully limits the rate of reaction under all conditions tested. In the
case of protonated linoleic acid, a number of steps are partially rate-limiting
at room temperature; three distinct mechanistic steps which include substrate
binding, an H2O/D2O sensitive step, and C-H bond cleavage have been
characterized. Use of glucose as a solvent viscosogen demonstrates that
substrate binding is approximately 48% rate-limiting for LA at 20 degrees C.
SBL-1 is one of the few enzymes that fit the definition of a "perfect enzyme",
in the sense that further optimization of any single step at room temperature
will not significantly increase the overall rate. At lower temperatures, the
step sensitive to solvent deuteration begins to dominate the reaction, whereas
at higher temperatures, the hydrogen abstraction step is rate-limiting. The pH
dependence of kcat/Km for SBL-1 can be explained as arising from two pKa's, one
controlling substrate binding and the other substrate release. Below pH 7.8, the
rate of substrate release increases, thus decreasing the commitment to catalysis
and unmasking the large intrinsic isotope effect on the subsequent hydrogen
abstraction. An abnormally high pKa, in the range of 7-8, has been determined
for LA in the concentration range employed in these studies. We propose that the
negatively charged form of LA, predominating above pH 7.8, is the preferred
substrate with larger commitments to catalysis.
|
