Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A

Sukalyan Chatterjee, Dominic Suciu, Ross E. Dalbey, Peter C. Kahn, Masayori Inouye

Research output: Contribution to journalEditorialpeer-review

39 Scopus citations

Abstract

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 Km of signal peptidase I was 0.0165 mM. The kcat was 8.73 s-1. The Km 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, kcat, is two to four orders of magnitude greater as well. Thus, the specificity constant, kcat/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.

Original languageEnglish (US)
Pages (from-to)311-314
Number of pages4
JournalJournal of molecular biology
Volume245
Issue number4
DOIs
StatePublished - Jan 1 1995

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • Escherichia coli
  • OmpA signal peptide
  • Secretion
  • Signal peptidase I
  • Staphylococcal nuclease A

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