In the final set of experiments

In the final set of experiments, the dependence of the reaction rate on viscosity was determined. The experimental protocol previously used to study the reaction of LOX with AA was used., Reactions of 5-LOX and AA were carried out at different relative viscosities in Tris buffer (25mM, pH 8.0) at 20°C. Buffer solutions of 0%, 14.0%, 24.0% and 32% by weight sucrose were prepared corresponding to relative viscosities of 1, 1.5, 2.2 and 3.2, respectively, at 20°C. The AA concentrations used ranged from 5 to 40μM, with 13-HPODE added to a final concentration of 2.5μM. The reactions were monitored at 235nm and initial rates were calculated. The decrease in with increased solvent viscosity suggests a diffusion-controlled rate-limiting step. For a completely diffusion-controlled reaction, the dependence of the rates on relative viscosity has a slope of 1. At 20°C, the rate of Palosuran of AA by 5-LOX is 60±4% diffusion-controlled for (slope of 0.61, A). Since AA and 7,7--AA are identical substrates, the observed difference in their rates should be due solely to differences in their chemical step. Therefore, we used 7,7--AA as a slow substrate control, an essential contingent of viscosity studies. The reaction of 5-LOX with 7,7--AA (38% diffusion-controlled for ) is essentially independent of the viscosity of the solution (B), indicating that the enzyme is free from any inhibitory effect of sucrose. This comparison of AA and 7,7--AA allows us to conclude that the effect of viscosity is on binding/release steps and not the chemical step. Moreover there is very slight increase in observed even in higher viscosity conditions (38μM for AA at 32% sucrose), suggesting that the association of enzyme and substrate is rarely rate limiting for /. The solvent viscosity effects on / seem to be independent in case of either substrate (labeled and unlabeled). This suggests a possibility of conformational change or slow product release after the H-abstraction step. In the above context, comparison of the primary coordination sphere of iron in LOXs shows that 5-LOX active site is similar to sLOX-1. This intrigued us to investigate if both these LOXs share similar mechanisms, in spite of having striking differences in the enzymatic rates and isotope effects. It was realized that being more sterically hindered, 5-LOX requires extensive active site dynamics in order to accommodate the long substrate, AA (C20) as compared to LA (C18) in sLOX-1. However, presence of bulky residues (F421, F177), non-aromatic (L368) and conformationally flexible residues (L414, Q363) might hinder the process of substrate rearrangement leading to the observed lower reaction rates or isotope effects and abrogating the specificity of product formation, resulting in 8-HETE formation due to lack of π–π interaction. Similar studies have been reported for the role of I553 and T259 in sLOX-1, and F414 and R402 in 15-LOX (). Thus, we propose that enzyme dynamics might influence donor–acceptor distances, influencing the overall kinetics and/or extent of tunneling involved. In summary, we have measured non-competitive equilibrium kinetic isotope effects of 5-LOX using its physiological substrate, AA and its labeled analogue, 7,7--AA. Using appropriately labeled AA substrate in this work, a KIE on of ∼6 was observed in reactions of 5-LOX. 5-LOX normally displays a preference for C7-abstraction, but when presented with 7,7--arachidonic acid, the isotope effect on hydrogen atom abstraction resulted in ∼6 times increased abstraction from C10. The KIE increased to ∼20 on and 17 on /, when the enzyme reactions were corrected for 8-HETE formation (). Indeed, analysis of the product distribution revealed that 5-LOX displays a reversal of selectivity to bypass abstracting the deuterium atom from C7. The observed outcome is another example of isotope sensitive branching, where product distribution is altered manifold by isotopic substitution, responsible for decreasing the rate of the branch point step of reaction. The observation that the but not the / is affected by a change in viscosity suggests that the reaction rate seems to be restricted by the product release or associated conformational changes as seen in the parameter. Also, the relatively large KIE observed at / as well as non-significant change in values provides us an opportunity to speculate that the reaction is majorly dependent on rate of H-abstraction. Thus, the data overall shows that the chemistry is rate determining for the catalytic reaction using the parameter / although the catalytic step governing seems to be more complicated and involves other associated steps such as conformational changes or slow product release. To be certain about the extent of commitment factors, further studies needs to be done. A slight temperature dependence of rates observed in this work with AA raises the question whether the hydrogen atom transfer/PCET step involves quantum mechanical tunneling. Also, it provides an opportunity to investigate if there is mutual cooperative dynamics of AA and 5-LOX influencing the extent of tunneling involved or if there is independent movement of enzyme with substrate/product to ease out the H-abstraction from C10 instead of C7., The bulkier amino acids; F421, F177, L414, Q363 and L368, are suggested to be responsible for the observed reduced rate and altered specificity in 5-LOX, corresponding to those in sLO-1. The results described here lead us to show that 5-LOX shows a broader aspect of KIE for LOXs, along with a new formalism for the interpretation of isotope effects in enzyme-catalyzed H-transfers where the / parameter is critical in describing KIE than .