EFFECT OF MODIFICATION OF ARGININE RESIDUES ON THE ACTIVITY OF SOYBEAN LIPOXYGENASE-1

Arginine residues of soybean lipoxygenase-l was modified with an arginine-directed chemical modifier, 2,3-butanedione. Although inactivation was not visible if the enzyme reaction was monitored under the standard assay condition (83.3 mu M linoleic acid dispersed in 200 mM sodium berate, pH 9.0), rapid inactivation was observed with 5 mM sodium berate, pH 8.0. The inactivation was protected by the addition of a substrate, linoleic acid, in the modification mixture. Kinetic analyses indicated that one arginine residue accounted for the inactivation. Enzymological analyses showed that the modification narrowed the pH-activity profile of L-1 and made L-1 sensitive to salt concentration of the assay solution. Strong inactivation by modification was found at low salt concentration and low pH. This was not due to a physical change of the linoleic acid. On the other hand, product specificity of L-1 was not altered after modification. Taken together, the modified arginine residue(s) was thought to be not essential to the catalysis but have an important role in supporting an ideal electrostatic interaction within L-1 and/or between L-1 and a substrate even in sub-optimal reaction conditions.