Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A.
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.
Chatterjee
S
S
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
Suciu
D
D
Dalbey
R E
RE
Kahn
P C
PC
Inouye
M
M
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; C057184:outer membrane protein A precursor (E coli); D008836:Micrococcal Nuclease; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase