DETERMINATION OF K-M AND K(CAT) FOR SIGNAL PEPTIDASE I USING A FULL LENGTH SECRETORY PRECURSOR, PRO-OMPA-NUCLEASE A

摘要

An effective method for the determination of the activity of signal peptidase I (SPase I) of Escherichia coli is established using the hybrid protein pro-OmpA-nuclease A as substrate. Pro-OmpA-nuclease A, a hybrid secretory precursor was purified to homogeneity under denaturing conditions. When this protein was refolded, it could be quantitatively processed by purified SPase I. The K-m of signal peptidase I was 0.0165 mM. The k(cat) was 8.73 s(-1). The K-m is 50 to 100 times lower than that obtained with peptide substrates indicating that SPase I has a significantly greater affinity for the protein substrate. The turnover number, k(cat), is two to four orders of magnitude greater as well. Thus, the specificity constant, k(cat)/(Km) is six orders of magnitude greater with pro-OmpA-nuclease A than with peptide substrates. This is the first determination of kinetics of SPase I with a protein substrate. [References: 17]