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PMID:7837264

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Citation

Chatterjee, S, Suciu, D, Dalbey, RE, Kahn, PC and Inouye, M (1995) Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A. J. Mol. Biol. 245:311-4

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.

Links

PubMed

Keywords

Amino Acid Sequence; Bacterial Outer Membrane Proteins/metabolism; Endopeptidases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Hydrolysis; Kinetics; Membrane Proteins; Micrococcal Nuclease/metabolism; Molecular Sequence Data; Protein Precursors/metabolism; Protein Processing, Post-Translational; Serine Endopeptidases

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:LEP

involved_in

GO:0006465: signal peptide processing

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:LEP

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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