Catalyzing Racemizations in the Absence of a Cofactor: The Reaction Mechanism in Proline Racemase

Abstract

The origin of the catalytic proficiency of the cofactor-independent enzyme proline racemase (ProR) has been investigated by a combined classical and quantum simulation approach with a hybrid quantum mechanics/molecular mechanics potential energy surface. The present study shows that the ProR reaction mechanism is asynchronous concerted with no distinct intermediate. Various mechanisms are investigated, and it is concluded that active site residues other than the Cys dyad are not involved in chemical catalysis. When compared to an analogous aqueous solution-phase reaction, we find that the free-energy barrier is reduced by 14 kcal/mol in ProR, although the reaction mechanisms in the enzyme and in water are similar. The computed catalytic effect is comparable to that in the isofunctional enzyme alanine racemase (AlaR). However, in AlaR the catalytic burden is divided between the cofactor pyridoxal 5'-phosphate and the enzyme environment, whereas in ProR it is borne entirely by the enzyme environment. This is ascribed to a highly preorganized active site facilitating transition state stabilization via a tight network of hydrogen bonds donated by nearby active site residues.

Engineering the Promiscuous Racemase Activity of an Arylmalonate Decarboxylase

“…[5,6] Cofactor-indepen-A C H T U N G T R E N N U N G dent amino acid racemases are essential enzymes of pathogenic organisms and, thus, potential targets for the treatment of infectious diseases. [7] Consequently, their molecular mechanism has received much attention in the last few years. Recent calculations for glutamate racemase (GluR) [8] and proline racemase (ProR) [7] suggest that the deprotonation and protonation is done simultaneously by two cysteines situated in an opposite orientation in the active site (Scheme 1 b, path b2).…”

Substituent Effects on Carbon Acidity in Aqueous Solution and at Enzyme Active Sites

“…55 This proposed effect of such hydrophobic active sites on reactivity provide a simple rationale for the otherwise difficult to fathom rate accelerations for substrate deprotonation. This proposal has not been strongly criticized, nor is it universally accepted, 47, 56 perhaps because of the difficulty in accepting this passive role for exceptionally active enzymes, in achieving their large catalytic rate accelerations.…”

Multiscale Quantum‐Classical Simulations of Enzymes

“…This may be achieved for SE methods within the so-called specific reaction parameter (SRP) approach introduced by Truhlar and Rossi [44] or in the empirical valence bond approach (EVB) of Warshel and Weiss. [45] We have employed DFT, [23,24] traditional SE methods, [33,46] and SE-SRP approaches [9,[47][48][49][50][51][52] to study enzymatic reactions. The guiding principle in choosing the PES has been to employ the cheapest possible method, without sacrificing the goal of near chemical accuracy.…”

“…[31] This greatly improved the accuracy of the computed KIE, and has been used extensively since. [9,29,33,34,46,48,51,52,82,83] Mass-perturbation techniques for PI KIE have also been adopted by others in the study of small molecules. [84][85] To further enhance the efficiency of the PI simulations, one may use higher order factorizations of the DM operator.…”