Comparative kinetic isotope effects on first- and second-order rate constants of soybean lipoxygenase variants uncover a substrate-binding network

Abstract

Edited by Ruma Banerjee Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first-and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O 2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate.

Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases

“…Importantly, the D k cat /K M value was seen to rise in a hyperbolic fashion with increasing OS concentration ( Figure 7A). A dissociation constant, K d , for OS binding to SLO-1 could be estimated from these kinetic assays as 0.6 ± 0. several large aliphatic active site residues to smaller volume side chains along the putative substrate binding channel gave rise to increased off rates for substrate binding [99]. The aggregate data support that substrate binding is regulated by a two-step mechanism involving the initial binding and a substrate reorientation within the active site.…”

“…These effects translate to a fully rate-limiting C-H cleavage step for all temperatures in the presence of allosteric effector. Analogous to the effect of OS, strategic site-directed mutagenesis of several large aliphatic active site residues to smaller volume side chains along the putative substrate binding channel gave rise to increased off rates for substrate binding [99]. The aggregate data support that substrate binding is regulated by a two-step mechanism involving the initial binding and a substrate reorientation within the active site.…”

“…From this analysis, OS addition decreases the commitment factor for catalysis (i.e., lowers Figure 6. Kinetic isotope effects reported for the first-order ( D k cat ) and second-order ( D k cat /K M ) rate constants for the reaction of SLO with substrate LA, as a function of temperature (A) [99]. (B) Schematic representation of the reaction coordinate for SLO reaction with LA at 30 • C (solid line).…”

“…Below room temperature, the value of D k cat /K M drops [ 89 ]. At 5 °C, the D k cat /K M for the LA reaction with SLO-1 is 7.7 ± 1.7, whereas the D k cat is 67 ± 5 at the same temperature ( Figure 6 A) [ 99 ]. The observation of D k cat/K M << D k cat under these conditions implicates an additional (partial) rate-limiting step(s) preceding the C-H cleavage step.…”

“…The observation of D k cat/K M << D k cat under these conditions implicates an additional (partial) rate-limiting step(s) preceding the C-H cleavage step. The former has been recently attributed to a combination of substrate binding and a partially rate-limiting isomerization after substrate binds and before C-H bond cleavage [ 89 , 99 ]. Similar trends have been characterized for h15-LOX-1 [ 81 , 97 ], suggesting a potentially conserved regulatory mechanism of the rate-limiting step(s) of LOX chemistry in higher eukaryotes.…”