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Volume 272, Number 50, Issue of December 12, 1997
pp. 31293-31300
(Received for publication, August 18, 1997, and in revised form, October 2, 1997)
From the Howard Hughes Medical Institute and the Departments of
Medicine and of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, Missouri 63110
Enteropeptidase, also known as
enterokinase, initiates the activation of pancreatic hydrolases by
cleaving and activating trypsinogen. Enteropeptidase is synthesized as
a single-chain protein, whereas purified enteropeptidase contains a
Bovine Proenteropeptidase Is Activated by Trypsin, and the
Specificity of Enteropeptidase Depends on the Heavy Chain
47-kDa serine protease domain (light chain) and a disulfide-linked
120-kDa heavy chain. The heavy chain contains an amino-terminal
membrane-spanning segment and several repeated structural motifs of
unknown function. To study the role of heavy chain motifs in substrate
recognition, secreted variants of recombinant bovine proenteropeptidase
were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the
transmembrane domain) and the catalytic light chain (pro-HL-BEK (where
BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low
catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified
enteropeptidase from bovine intestine (Km = 5.6 µM and kcat = 4.0 s
1) and by HL-BEK (Km = 5.6 µM and kcat = 2.2 s1), but not by L-BEK (Km = 133 µM and kcat = 0.1 s1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold
greater catalytic efficiency than did L-BEK. Qualitatively similar
results were obtained at pH 8.4. In contrast to this striking
difference in trypsinogen recognition, the small synthetic substrate
Gly-Asp-Asp-Asp-Asp-Lys-
-naphthylamide was cleaved with similar
kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s1). The presence of the heavy chain also influenced the
rate of reaction with protease inhibitors. Bovine pancreatic trypsin
inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM)
over L-BEK (Ki = 698 nM and
Ki* = 6.2 nM). Soybean trypsin
inhibitor exhibited a reciprocal pattern, inhibiting L-BEK
(Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little
influence on the recognition of small peptides, but strongly influences
macromolecular substrate recognition and inhibitor specificity.
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