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, Bo th chimeras exhibited the characteristic features of aspartic proteinases, such as auto-activation at low pH and abrogation of enzymatic activity by p epstatin. The activity of the chimera containing the N-terminal domain of r hizopuspepsinogen 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 dom ain of porcine pepsinogen and the C-terminal domain of rhizopuspepsinogen e xhibited catalytic activity, measurable by a spectrophotometric assay. Kine tic data and inhibitor analyses strongly suggest that interdependency may e xist between adjacent subsites contributed by different domains. Therefore, in order to develop an optimal substrate or inhibitor, the effect of adjac ent residues of the ligand has to be examined along with the preferences fo r each subsite. (C) 2000 Academic Press.