Structural enzymology, ligand recognition and mechanistic pathways

A rational approach to drug design involves a better understanding of drug-receptor interactions in order to specify different forces involved in binding interactions and molecular recognition process. This is particularly important in addressing specificity. For the purpose of this thesis, an azapeptide inhibitor JM2-73 of the cysteine protease papain (EC 3.4.22.2) was designed based on the affinity of the S2 subsite of papain for a large hydrophobic residue. A complex of papain with the inhibitor JM2-73 which was to deliver the azaglycine carbonyl to the active-site cysteine was studied by X-ray crystallography. The structural studies carried out on the enzyme-inhibitor complex reveal the observed mode of binding of an azapeptide inhibitor within the active site of papain.; This thesis includes the determination of the three-dimensional structure of a number of active site mutants and possible enzyme-inhibitor complexes of mandelate racemase (MR; EC 5.1.2.2) from Pseudomonas putida that have helped in full elucidation of the enzyme mechanism and the domain movements that occur upon substrate binding.; This thesis describes the effect of such rational mutations carried out within the active site of MR in order to obtain the functionality of muconate lactonizing enzyme (MLE; EC 5.5.1.1). The results of the structural studies led to a better understanding of the relationship between the MR and MLE subfamilies of the enolase superfamily.; The β-ketoadipate pathway consists of two parallel branches where such enzymes, namely, MLE and 3-Carboxy-cis, cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.2) take part in analogous addition reactions using different mechanisms. While MLE carries out 1,4 addition reaction in a syn fashion, CMLE carries out similar reaction in anti manner. The two enzymes are not homologous to each other and their sequence alignments show that they are members of different superfamilies. The three-dimensional structure determination of CMLE shows that it has the overall fold of the fumarase superfamily members of which carry out the addition reaction in an anti fashion. Both of these enzymes belonging to different superfamilies have different mechanisms and have been confirmed to have different structures. The structure determination of CMLE has helped in establishing its place as the member of fumarase superfamily. (Abstract shortened by UMI.)...