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J Biol Chem, Vol. 273, Issue 35, 22672-22680, August 28, 1998
From the In the central and peripheral nervous
systems, the neuropeptide precursor proenkephalin must be
endoproteolytically cleaved by enzymes known as prohormone convertases
1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this
study, we have investigated the specificity of recombinant mouse PC2
for proenkephalin-related internally quenched (IQ) peptides, for
methylcoumarin amide-based fluorogenic peptides, and for recombinant
rat proenkephalin. IQ peptides exhibited specificity constants
(kcat/Km) between 9.4 × 104 M
Specificity of Prohormone Convertase 2 on Proenkephalin and
Proenkephalin-related Substrates
,
,,
Department of Biochemistry and Molecular
Biology, Louisiana State University Medical Center, School of
Medicine, New Orleans, Louisiana 70112, the § Department
of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020, Sao Paulo, Brazil, the Neuropeptide Structure and
Metabolism Laboratory, Institut de Recherches Cliniques de
Montreal, Montreal, Quebec, Canada 2W 1R7, and the ** Howard Hughes
Medical Institute and Department of Biochemistry, University of Chicago
Medical School, Chicago, Illinois 60637
1 s1
(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where
Abz is ortho-aminobenzoic acid and EDDnp is
N-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 × 104 M1 s1
(Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the
peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is
met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino
acids were hydrolyzed with specificity constants ranging between
2.0 × 103 M1
s1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin
amide) and 1.8 × 104 M1
s1 (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is
pyroglutamic acid). Substrates containing only a single basic residue
were not appreciably hydrolyzed, and substrates lacking a P4 Arg
exhibited kcat of less than 0.05 s1. Substitution of ornithine for Lys at the P4 position
did not significantly affect the kcat but
increased the Km 2-fold. Data from both sets of
fluorogenic substrates supported the contribution of a P4 Arg to PC2
preference. Analysis of proenkephalin reaction products using
immunoblotting and gel permeation chromatography demonstrated that PC2
can directly cleave proenkephalin and that the generation of small
opioid peptides from intermediates is mediated almost entirely by PC2
rather than by PC1. These results are in accord with the analysis of
PC2 knock-out brains, in which the amounts of three mature enkephalins
were depleted by more than three-quarters.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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