Challenges and Advances in Validating Enzyme Design Proposals: The Case of Kemp Eliminase Catalysis

Abstract

One of the fundamental challenges in biotechnology and biochemistry is the ability to design effective enzymes. Despite recent progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the preorganization of the environment, which is the key factor in enzyme catalysis. Thus, rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work considers the catalytic effects in different Kemp eliminases as a benchmark for a computer aided enzyme design. It is shown that the empirical valence bond provides a powerful screening tool, with significant advantage over current alternative strategies. The insights provided by the empirical valence bond calculations are discussed emphasizing the ability to analyze the difference between the linear free energy relationships obtained in solution to those found in the enzymes. We also point out the trade off between reliability and speed of the calculations and try to determine what it takes to obtain reliable computer aided screening.

Why nature really chose phosphate

“…Warshel et al 2006b and references cited therein), but also the effects that mutations are observed to have on the catalysis (as has been done in countless computational studies on a wide range of systems, see e.g. Frushicheva et al 2011 ;Liu & Warshel, 2007 ;Roca et al 2009 ;Shurki & Warshel, 2004). This is the only way one can not only characterize the mechanism of the enzyme-catalyzed reactions, but also understand the molecular basis for the observed effect.…”

“…Of course, the difference between our approach and the qualitative approach promoted by e.g. (Lassila et al 2011) can also be realized by the fact that we have been actually able to reproduce the effect of different mutations Frushicheva et al 2011 ;Liu & Warshel, 2007 ;Roca et al 2009 ;Shurki & Warshel, 2004), whereas (Lassila et al 2011) could only argue that they suggested the direction of the effect of the Arg finger, without being able to even hint at what the magnitude of those effects may be, and were also forced to invoke factors such as water positioning, thereby overlooking the key effect of the P-loop and the Mg 2+ ion, as well as the fact that catalysis by water positioning has long been shown to have only a minor catalytic effect (both as NAC and as an entropic effect, see discussion in Warshel et al 2006b and references cited therein).…”

Prechemistry barriers and checkpoints do not contribute to fidelity and catalysis as long as they are not rate limiting

“…So arguing that such approaches cannot predict the activity of altered enzymes is one of the most problematic parts of the SABW paper. The fact is that we have repeatedly predicted the activity of countless altered enzymes [24,[51][52][53], and we are not aware of any such prediction based on the dynamical concepts promoted by SABW and others. We would also like to clarify that it is easy to imply that all calculations are important in order to understand catalysis, and that this might be popular in some circles.…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…EVB is based on a semiempirical Hamiltonian which describes reactants and products with their resonance structure (explicitly defining atom connectivity). Although EVB energies are less accurate than ab initio and DFT QM/MM methods, free energy calculations are orders of magnitude quicker and still can achieve accurate results [183,184], making EVB a suitable tool to score a bigger number of mutants.…”