Purification and characterization of a novel plant aspartic proteinase: Elucidating the role of the plant specific sequence.

摘要

This thesis is a structure-function study of a novel plant aspartic proteinase (AP) and a chimeric AP, which includes a plant specific sequence (PSS). An AP was purified, partially characterized, and N-terminally sequenced from the leaves of Medicago sativa (L: alfalfa) (MsAP). The MsAP was monomeric, demonstrated a molecular weight of approximately 33.5 kDa, and catalyzed the cleavage of a broad spectrum of peptide bonds of hydrophobic amino acids over the pH range of 2.6 to 6.4. Using a synthetic substrate, the MsAP possessed a Km of 29.8 muM and kcat of 4.75 s-1 , and was inhibited by pepstatin. These characteristics were consistent with the properties of an AP. N-terminal amino acid sequencing indicated considerable similarity with the cyprosin (60% homology) and barley APs (50% homology).; The PSS region from the Arabidopsis thaliana AP was rationally redesigned into pepsinogen, a mammalian AP, at the position identical to that found in plant APs. This chimeric enzyme was over-expressed in Pichia pastoris and then activated to yield a functional pepsin-PSS chimera. The inclusion of the PSS domain resulted in increases in pH stability above 6 and temperature stability above 60°C compared to commercial pepsin. Differential scanning calorimetry of the chimeric enzyme illustrated an approximate 2°C increase in denaturation temperature (Tm) with an increase in cooperativity. Kinetic analysis indicated an increase in K m and decreased kcat compared with pepsin, but with a catalytic efficiency similar to a reported monomeric plant aspartic proteinase. Circular dichroism data showed an increase in alpha-helical content of the chimeric enzyme similar to that found for the PSS. Using the oxidized insulin B-chain, the chimeric enzyme demonstrated more restricted substrate specificity in comparison to commercial pepsin. An enzymatically deglycosylated form of the chimeric AP demonstrated reduced catalytic efficiency and alpha-helical content, while possessing similar substrate specificity, pH- and thermostability.