COMPARATIVE MODELING OF BARLEY-GRAIN ASPARTIC PROTEINASE - A STRUCTURAL RATIONALE FOR OBSERVED HYDROLYTIC SPECIFICITY

A model of the barley-grain aspartic proteinase (HvAP; Hordeum vulgare aspartic proteinase) has been constructed using the rule-based compar ative modelling approach encoded in the COMPOSER suite of computer pro grams. The model was based on the high resolution crystal structures o f six highly homologous aspartic proteinases. Results suggest that the overall three-dimensional structure of HvAP (excluding the plant-spec ific insert; 104 residues in HvAP) is closer to human cathepsin D than other aspartic proteinases of known three-dimensional structure. Comp arisons of the complexes with the substrate modelled in the active sit e of HvAP with those of the same substrate modelled in the active site of other aspartic proteinases of known three-dimensional structure an d specificity, define residues that may influence hydrolytic specifici ty of the barley enzyme. We have identified residues in the S-4 (Ala(1 2)), S-3 (Gln(13), Thr(111)), S-2 (Ala(222), Thr(287), Met(289)), S-1' and S-3' (Ile(291)), S-2' and S-3' (Gln(74)), S-2' (Arg(295)), and S- 3' (Pro(292)) pockets, that may account for the observed trends in the kinetic behaviour and specificity when compared to other aspartic pro teinases. The plant-specific inserted sequence, which may play a role in the transport of HvAP to plant vacuoles (lysosomes), is similar to the saposins and is predicted to be a mixed alpha-helical and beta-str and domain.