Protein structure to function: insights from computation.

Computation plays an important role in functional genomics. THEMATICS is a computational method that predicts chemical and electrostatic properties of residues in enzymes and utilizes information contained in those predictions to identify active sites. The only input required is the three-dimensional structure of the query protein. The identification of residues involved in catalysis and in recognition is discussed. The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes. In addition, Kex2 is specific for dibasic sites and THEMATICS finds the recognition residues for both the S1 and S2 sites of Kex2. In contrast, no such recognition sites are found for the non-specific enzyme subtilisin. The ability to identify sites that govern recognition opens the door to better understanding of specificity and to the design of highly specific inhibitors.

THEMATICS: a simple computational predictor of enzyme function from structure.

We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in proteins of known structure. Results are featured for three enzymes: triosephosphate isomerase (TIM), aldose reductase (AR), and phosphomannose isomerase (PMI). We note that TIM and AR have similar structures but catalyze different kinds of reactions, whereas TIM and PMI have different structures but catalyze similar reactions. Analysis of the theoretical microscopic titration curves for all of the ionizable residues of these proteins shows that a small fraction (3-7%) of the curves possess a flat region where the residue is partially protonated over a wide pH range. The preponderance of residues with such perturbed curves occur in the active site. Additional results are given in summary form to show the success of the method for proteins with a variety of different chemistries and structures.

A model for enzyme-substrate interaction in alanine racemase.

We report on a theoretical model for the complex of the enzyme alanine racemase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phosphate). Electrostatic potentials were calculated and ionization states were predicted for all of the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265' has an unusually low predicted pK(a) of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority of Tyr265' side groups will be in the phenolate form. This lends support to the experimental evidence that Tyr265' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predicted average charges of +0.91 and +0.14, respectively, at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alanine, and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able to act as a base. Cys311', which is located very close to the active site, has an unusually low predicted pK(a) of 5.8 and at pH 7.0 has a predicted average charge of -0.72. The very low predicted charge for Lys129 is consistent with experimental evidence that it is carbamylated, since an unprotonated amine group is available to act as a Lewis base and form the carbamate with CO(2). Repeating the pK(a) calculations on the enzyme with Lys129 in carbamylated form predicts trends similar to those of the uncarbamylated enzyme. It appears that the enzyme has the ability to stabilize negative charge in the region of the active site. Implications for selective inhibitor design are discussed.