Computer simulations of the catalytic mechanism of wild-type and mutant ß-phosphoglucomutase.

ß-Phosphoglucomutase (ß-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of ß-glucose-1-phosphate to ß-glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of ß-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

The alkaline hydrolysis of sulfonate esters: challenges in interpreting experimental and theoretical data.

Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., Org. Biomol. Chem. 10, 2012, 8095) presented a nonlinear Brønsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its N-oxide and N-methylpyridinium derivatives. Inclusion of these compounds converts the Brønsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data.

Synthesis and evaluation of analogues of N-phthaloyl-l-tryptophan (RG108) as inhibitors of DNA methyltransferase 1.

DNA methyltransferases (DNMT) are promising drug targets in cancer provided that new, more specific, and chemically stable inhibitors are discovered. Among the non-nucleoside DNMT inhibitors, N-phthaloyl-l-tryptophan 1 (RG108) was first identified as inhibitor of DNMT1. Here, 1 analogues were synthesized to understand its interaction with DNMT. The indole, carboxylate, and phthalimide moieties were modified. Homologated and conformationally constrained analogues were prepared. The latter were synthesized from prolinohomotryptophan derivatives through a methodology based amino-zinc-ene-enolate cyclization. All compounds were tested for their ability to inhibit DNMT1 in vitro. Among them, constrained compounds 16-18 and NPys derivatives 10-11 were found to be at least 10-fold more potent than the reference compound. The cytotoxicity on the tumor DU145 cell line of the most potent inhibitors was correlated to their inhibitory potency. Finally, docking studies were conducted in order to understand their binding mode. This study provides insights for the design of the next-generation of DNMT inhibitors.

Computational protein engineering: bridging the gap between rational design and laboratory evolution.

Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation "hotspots" with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies.