On electrostatic effects, minimal motion and other catalytic strategies used by enzymes

Enzymes are powerful biocatalysts that provide rate accelerations of up to 1019 fold compared to the corresponding uncatalyzed reaction in solution. The origin of the remarkable performance displayed by enzymes has fascinated and puzzled researchers for over a hundred years. It is clear that the catalytic effect is a consequence of the higher degree of transition state stabilization for the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction. It is still not well understood exactly how this transition state stabilization occurs and the relative importance of various catalytic effects are discussed. Catalytic effects involving electrostatics, near attack conformers, dynamic effects and an economy in atomic motion are discussed in this thesis. The importance of electrostatic effects is corroborated in this thesis. A single hydrogen bond in transition state constitutes an important difference between amidases and esterases. A hydrogen bond in transition state is found in all sixteen analyzed amidases representing ten different reaction mechanisms and eleven different folding families. The hydrogen bond is shown to be either substrate assisted or enzyme assisted. The role of this hydrogen bond is to assist nitrogen inversion in amidases. Esterases lack this interaction in transition state and therefore they are very poor catalysts in the hydrolysis of amides. Electrostatic interactions are found to facilitate proton transfer that enhances the rate of lipase catalyzed N-acylation of amino alcohols. In this thesis electrostatic effects in the substrate are shown to be important for the lipase catalyzed transacylation of acrylates The α,β-double bond present in acrylates introduce electronic effects that has the consequence of restricting the conformational freedom of the substrate in its ground state to two flat conformations, s-cis and s-trans. It is shown that acrylates form near attack conformers (NACs) from their ground state s-cis/s-trans planar conformations. The ability of the enzyme to accommodate such apparent s-cis/s-trans substrate conformations dictates the probability to form productive transition states and thus the reaction rate. Dynamic effects are important in enzymes. In this thesis it is found that a point mutation increases the flexibility of a neighbouring residue in Candida antarctica lipase B. This allows the mutated enzyme to explore conformations not accessible for the wild-type enzyme. The dynamics has the effect to decrease steric interactions in transition state with concomitant rate increase for the transacylation of methyl methacrylate. In this thesis an economy of atomic motion during enzyme catalysis is observed. Nitrogen inversion in amidases constitutes an interesting example. A rotation as part of the reaction mechanism for amide bond hydrolysis would involve much more motion.QC 2011051