The processing of proinsulin is being used in our laboratory to examine the selectivity demonstrated by propeptide converting enzymes. PC1 is responsible for the cleavage of the B-chain/Cpeptide junction in proinsulin. We have recently succeeded in generating recombinant human proinsulin (hPI) from bacteria, and PC1 using a baculovirus system. We have also developed an in vitro assay and are now attempting to determine kinetic constants for the action of a converting enzyme with its authentic substrate. Our initial results indicate that the Km for hPI is approximately 2 iiM compared to 25 nM for Pyr-RTKR-AMC. In the absence of an active site directed probe, RIA determination of enzyme concentration provides a lower limit for /tcat of 1 s-' for hPI and 0.5 S'1 for PyrRTKR-AMC. From this, the ratios of the specificity constants C<cat/Km) for these two substrates indicate a 25-fold preference of PC1 for hPI as substrate compared to Pyr-RTKR-AMC. Taken together with our recent observation that chemical modification of hPI alters its processing by PC1, these results suggest that substrate recognition by propeptide converting enzymes involves elements of the three-dimensional structure of the propeptide. (Funded by the Health Future Foundation, the Nebraska Cancer and Smoking-related Disease Program, and NIH/NIDDK.).
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology