J. Bryan Jones

Glycomethanethiosulfonates: powerful reagents for protein glycosylation

Abstract Twelve novel glycomethanethiosulfonate (glyco-MTS) protein glycosylation reagents have been prepared. Their use in a controlled site-selective glycosylation strategy that combines site-directed mutagenesis with chemical modification allows protein glycosylation with concomitant control of (i) site, (ii) carbohydrate, (iii) anomeric stereochemistry, (iv) sugar to protein spacer arm nature and (v) degree of glycan protection. The ability of these highly selective and yet reactive reagents has been illustrated by the introduction of d -glucosyl and N-Ac- d -glucosaminyl residues to both external and hindered internal sites in a model protein — the serine protease enzyme subtilisin Bacillus lentus (SBL) — using corresponding gluco-MTS 1 and N-Ac-glucosamine-MTS 2. Molecular modelling studies provide a rationale for the strikingly different effects of these reagents on the properties of the protein despite differing only in the nature of their C-2 substituents.

Structure-based design of a potent transition state analogue for TEM-1 β-lactamase

The structure of the plasmid-mediated β-lactamase TEM-1 has been solved in complex with a designed boronic acid inhibitor (1R)-1-acetamido-2-(3-carboxyphenyl)ethane boronic acid at 1.7 Å resolution. The boronate inhibitor was designed based on the crystallographic coordinates of the acyl-enzyme intermediate of TEM-1 bound to the substrate penicillin G. The boronate–TEM-1 complex is highly ordered and defines a novel transition state analogue of the deacylation step in the β-lactamase reaction pathway. The design principles of this highly effective inhibitor (Ki=110 nM) and the resulting structural and mechanistic implications are presented.

Dilemma regarding an active site model for porcine pancreatic lipase

Abstract An active-site model for porcine pancreatic lipase (PPL) is described that focuses further attention on the factors determining PPL-specificity in order to facilitate the development of a comprehensive active site model that will be universally applicable in all asymmetric synthetic applications of the enzyme.

A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase

Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.

Enzymes in organic synthesis 50. Probing the dimensions of the large hydrophobic binding region of the active site of pig liver esterase using substituted aryl malonate substrates

Abstract The active site model reported recently for the synthetically useful enzyme pig liver esterase (PILE) permits the structural specificity and stereoselectivity of the enzyme to be interpreted and predicted for a wide range of substrates. The specifications of the dimensions of this model were based on the specificity data available at that time. In order to test the model further, and to delineate more accurately the dimensions of its large hydrophobic (H L ) binding pocket, PLE-catalyzed hydrolyses of dimethyl ortho- and para-methyl-, ethyl-, isopropyl-, and tert-butylphenyl 2-methylmalonate substrates have been carried out. Each of these malonate diesters proved to be a good substrate of the enzyme. In every case, the pro-S ester group was hydrolyzed to give R-acid-ester products of 78−>-97% ee. The results show that the initial volume specified for the HL pocket was too small to accommodate the larger aryl groups of this substrate series. A modified model with an appropriately enlarged H L region is presented. The sizes of the other binding pockets remain unchanged.

Probing the specificity of the S1 binding site of M222 mutants of subtilisin B. lentus with boronic acid inhibitors

Abstract Specificity differences between the S1-pockets of subtilisin B. lentus (SBL), and its M222C/S mutants have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenylboronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both the M222C and M222S enzymes. Specificity differences between the S 1-pockets of subtilisin from B. lentus (SBL), and its M222C/S mutants, have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenyl boronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both M222C and M222S enzymes.

Expanded structural and stereospecificity in peptide synthesis with chemically modified mutants of subtilisin

Abstract Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d -amino acid acyl donors, generating dipeptides containing d -Phe, d -Ala and d -Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l -Ala-NH 2 as acyl acceptors in their S 1 ′ pockets, which WT-SBL will not.

Probing the specificity of the S1 binding site of subtilisin Carlsberg with boronic acids

Abstract A range of aryl and arylalkyl boronic acids has been prepared and evaluated as inhibitors of the serine protease subtilisin Carlsberg, with the goal of exploring the factors controlling binding to the S 1 site.

Enzymes in organic synthesis 51. Probing the dimensions of the large hydrophobic pocket of the active site of pig liver esterase

Abstract The dimensions of the large hydrophobic pocket (H L ) of the active site model of pig liver esterase (PLE) were probed using a series of aliphatic and phenylic malonates. Results from the hydrolyses of these new unnatural substrates permitted the extension of the H( L ) pocket to give the new dimensions of 6.2 x 2.3 x 3.9 A for a total volume of

Altering the specificity of subtilisin B. Lentus by combining site-directed mutagenesis and chemical modification

56 A 3 .