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

doi:10.1006/jmbi.1994.0025

Determination of K_m and k_cat 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 journal › Editorial › peer-review

42 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 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/K_m 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 language	English (US)
Pages (from-to)	311-314
Number of pages	4
Journal	Journal of molecular biology
Volume	245
Issue number	4
DOIs	https://doi.org/10.1006/jmbi.1994.0025
State	Published - 1995

All Science Journal Classification (ASJC) codes

Structural Biology
Molecular Biology

Keywords

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

Access to Document

10.1006/jmbi.1994.0025

Cite this

@article{984af0382d934d2885bda058c0dce8ba,

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

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.",

keywords = "Escherichia coli, OmpA signal peptide, Secretion, Signal peptidase I, Staphylococcal nuclease A",

author = "Sukalyan Chatterjee and Dominic Suciu and Dalbey, \{Ross E.\} and Kahn, \{Peter C.\} and Masayori Inouye",

note = "Funding Information: This work was supported by a grant from the American Cancer Society (ACS grant MV-499Q). We also acknowledge support from the Center for Advance Food Technology (CAFT) and the New Jersey Agricultural Experiment Station (paper nos. D-01405-93-1 and D10535-93-11). CAFT is a New Jersey Commission on Science and Technology Center. R.E.D. is grateful to the National Science Foundation (DCB-9020759) for supporting this work. We also thank Dr Mark Lively and Mohammed Nusier of Wake Forest University, The Bowman Gray School of Medicine, for the N-terminal sequence determination of the cleaved product of pro-OmpA-nuclease.",

year = "1995",

doi = "10.1006/jmbi.1994.0025",

language = "English (US)",

volume = "245",

pages = "311--314",

journal = "Journal of molecular biology",

issn = "0022-2836",

publisher = "Academic Press",

number = "4",

}

TY - JOUR

T1 - Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A

AU - Chatterjee, Sukalyan

AU - Suciu, Dominic

AU - Dalbey, Ross E.

AU - Kahn, Peter C.

AU - Inouye, Masayori

N1 - Funding Information: This work was supported by a grant from the American Cancer Society (ACS grant MV-499Q). We also acknowledge support from the Center for Advance Food Technology (CAFT) and the New Jersey Agricultural Experiment Station (paper nos. D-01405-93-1 and D10535-93-11). CAFT is a New Jersey Commission on Science and Technology Center. R.E.D. is grateful to the National Science Foundation (DCB-9020759) for supporting this work. We also thank Dr Mark Lively and Mohammed Nusier of Wake Forest University, The Bowman Gray School of Medicine, for the N-terminal sequence determination of the cleaved product of pro-OmpA-nuclease.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

KW - Escherichia coli

KW - OmpA signal peptide

KW - Secretion

KW - Signal peptidase I

KW - Staphylococcal nuclease A

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DO - 10.1006/jmbi.1994.0025

M3 - Editorial

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VL - 245

SP - 311

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JO - Journal of molecular biology

JF - Journal of molecular biology

IS - 4

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