Chimeric aspartic proteinases and active site binding

Two chimeric enzymes were constructed by exchanging domains between porcine pepsinogen and rhizopuspepsinogen in order to examine the contributions of the subsites present on different domains toward enzymatic specificity. Both chimeras exhibited the characteristic features of aspartic proteinases, such as auto-activation at low pH and abrogation of enzymatic activity by pepstatin. The activity of the chimera containing the N-terminal domain of rhizopuspepsinogen and the C-terminal domain of porcine pepsinogen (rhzNppC) could be observed by HPLC after prolonged incubation with the substrates. In contrast, the reciprocal chimera, ppNrhzC, containing the N-terminal domain of porcine pepsinogen and the C-terminal domain of rhizopuspepsinogen exhibited catalytic activity, measurable by a spectrophotometric assay. Kinetic data and inhibitor analyses strongly suggest that interdependency may exist between adjacent subsites contributed by different domains. Therefore, in order to develop an optimal substrate or inhibitor, the effect of adjacent residues of the ligand has to be examined along with the preferences for each subsite.