Seine Peptidase Analysis
Structural Analysis of Serine Peptidase-Inhibitor Complexes
When analyzing these structures, we observed a reasonably strong hydrogen bond interaction (distance ~ NVL ≫ AAoF ≈ NVoL. Surprisingly, the unmodified AAF compound showed a substantially higher level of activity over the unmodified NVL compound. Initially, we hypothesized that the modified compounds (No H-Bond) would be less active. To our surprise, both modified compounds, AAoF and NVoL, showed negligible activity (Figure 16). Additionally, as [α-Chymotrypsin] was increased serially, 0.5, 1, 2, 5 (nM), the activity (RFUs), using AAF as the substrate, increased in a proportional manner (Figure 20).
SGPB is active on AAF and NVL but not AAoF or NVoL
When comparing the 4 tri-peptide …show more content…
Therefore, post-assay samples were sent for Mass Spectrometry analysis. The Mass Spectrometry analysis (High Resolution Electro-Spray Ionization Mass Spectrometry, HR-MS) came back positive with a clear peak (retention time = 5.172, m/z = 508.3) which correlates well with a [M+H]+ ion for the oxy-analogue (AAoF). Thus, it is apparent that this compound is stable in solution which leads to our conclusion that it is not cleaved by either enzyme, α-Chymotrypsin or …show more content…
Inhibition was not observed. Therefore, we hypothesized that this occurred because the binding ability of the AAoF compound is reduced compared to the unmodified AAF compound. We believe this to be due to the abrogation of the nucleophile-P1 amide hydrogen bond in the enzyme-substrate complex. Subsequently, we performed a broad range inhibition assay by increasing AAoF while keeping AAF constant. An expected trend was seen where the higher the AAoF concentration, the less activity (RFUs) observed. For AAF, we were able to estimate a Ki value of 0.772 mM (Figure 19,
When analyzing these structures, we observed a reasonably strong hydrogen bond interaction (distance ~ NVL ≫ AAoF ≈ NVoL. Surprisingly, the unmodified AAF compound showed a substantially higher level of activity over the unmodified NVL compound. Initially, we hypothesized that the modified compounds (No H-Bond) would be less active. To our surprise, both modified compounds, AAoF and NVoL, showed negligible activity (Figure 16). Additionally, as [α-Chymotrypsin] was increased serially, 0.5, 1, 2, 5 (nM), the activity (RFUs), using AAF as the substrate, increased in a proportional manner (Figure 20).
SGPB is active on AAF and NVL but not AAoF or NVoL
When comparing the 4 tri-peptide …show more content…
Therefore, post-assay samples were sent for Mass Spectrometry analysis. The Mass Spectrometry analysis (High Resolution Electro-Spray Ionization Mass Spectrometry, HR-MS) came back positive with a clear peak (retention time = 5.172, m/z = 508.3) which correlates well with a [M+H]+ ion for the oxy-analogue (AAoF). Thus, it is apparent that this compound is stable in solution which leads to our conclusion that it is not cleaved by either enzyme, α-Chymotrypsin or …show more content…
Inhibition was not observed. Therefore, we hypothesized that this occurred because the binding ability of the AAoF compound is reduced compared to the unmodified AAF compound. We believe this to be due to the abrogation of the nucleophile-P1 amide hydrogen bond in the enzyme-substrate complex. Subsequently, we performed a broad range inhibition assay by increasing AAoF while keeping AAF constant. An expected trend was seen where the higher the AAoF concentration, the less activity (RFUs) observed. For AAF, we were able to estimate a Ki value of 0.772 mM (Figure 19,