A THEORETICAL-STUDY OF THE ACTIVE-SITES OF PAPAIN AND S195C RAT TRYPSIN - IMPLICATIONS FOR THE LOW REACTIVITY OF MUTANT SERINE PROTEINASES

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residu es, Asp...His...Ser. The hydroxyl group of serine functions as a nucle ophile and the imidazole ring of histidine functions as a general acid /general base during catalysis. Similarly, the active site of the cyst eine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to e xist as a zwitterion (Asn...His(+)...Cys(-)) with the thiolate anion o f the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilis in and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock cal culations have been performed on the active sites of papain and the mu tant serine proteinase S195C rat trypsin. These calculations predict t hat the active site of papain exists predominately as a zwitterion (Cy s(-)...His(+)...Asn). However, similar calculations on S195C rat tryps in demonstrate that the thiol mutant is unable to form a reactive thio late anion prior to catalysis. Furthermore, structural comparisons bet ween native papain and S195C rat trypsin have demonstrated that the sp atial juxtapositions of the triad residues have been inverted in the s erine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.