The sequence and reactive site of ecotin. A general inhibitor of pancreatic serine proteases from Escherichia coli.
Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unusual in its ability to inhibit chymotrypsin, trypsin, and elastase. To address the structural basis of its broad specificity, the gene for ecotin has been cloned and its sequence determined. A promoter of the 17-base pair spacing class was identified, and the probable transcriptional start site lies 18 base pairs upstream from a ribosome binding locus. The gene is followed by a series of conserved repetitive extragenic palindromic sequences. Ecotin has a signal peptide of 20 amino acids which confirms its periplasmic localization. Sequence analyses by Edman degradation and mass spectrometry confirmed 71% of the deduced protein sequence of calculated monomeric molecular mass 16,096 Da. Comparisons of the primary structure for the 142-amino acid protein with the major classes of serine protease inhibitors suggest that ecotin is a novel inhibitor. The reactive site of ecotin was determined to be Met84 for its complexes with chymotrypsin, trypsin, and elastase. The scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors.
McGrath
M E
ME
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0446.
Hines
W M
WM
Sakanari
J A
JA
Fletterick
R J
RJ
Craik
C S
CS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D033903:Periplasmic Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D010196:Pancreatic Elastase