Sandbox Reserved 712: Difference between revisions
From Proteopedia
Jump to navigationJump to search
| Line 28: | Line 28: | ||
=== Enzymatic analysis of recombinant PRs === | === Enzymatic analysis of recombinant PRs === | ||
Samples PR<sub>drv1</sub> to PR<sub>drv6</sub> have been cloned and expressed in ''E. coli'' | Samples PR<sub>drv1</sub> to PR<sub>drv6</sub> have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs. | ||
Despite there were many mutations the k<sub>cat</sub> values still were between 30 and 50% of the wild-type value. In contrast the K<sub>m</sub> values of the mutants were (mostly) four- to eightfold higher than the wild-type PR. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) | Despite there were many mutations the k<sub>cat</sub> values still were between 30 and 50% of the wild-type value. In contrast the K<sub>m</sub> values of the mutants were (mostly) four- to eightfold higher than the wild-type PR. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study])<ref name="Molecular" /> | ||
Inhibition constants were determined by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor. | |||
PR<sub>drv5</sub> - which only had a specific activity of 5% of the wild-type value - also shows a smaller difference in k<sub>i</sub> value for darunavir, even if it contains 20 mutations. Among those, some are responsible for cross-resistance to other PIs (L10I, L33F, M46L, I54V, A71V, V82T, I84V, L89V and L90M). | |||
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants] | [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants] | ||
