Abstract
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 x 10(4) M-1 s-1 (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 x 10(4) M-1 s-1 (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 x 10(3) M-1 s-1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s-1 (<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 s-1. 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.
Highlights
¶ Supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)
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
Previous reports from our laboratory using cell-based systems have provided support for the idea that PC1 is the chief enzyme concerned with the generation of intermediate-sized peptides from proenkephalin (PE) and that PC2 is mostly responsible for the production of small, bioactive opioid peptides [5, 6]
Summary
¶ Supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq). In agreement with the notion that PC2 expression is correlated with more complete processing of PE, antisense experiments have shown that PC2 is largely responsible for the processing of PE into smaller opioid peptides in Rin cells [6] These data imply that PC2 can cleave at a wider range of sites within PE than PC1; the structural factors that differentiate PC1 from PC2 cleavage sites remain unclear. Studies of recombinant PC2 cleavage on natural substrates include proglucagon [18, 19], cholecystokinin-33 [20], and prodynorphin [21] Comparative work on both enzymes includes reports on the cleavage of proneuropeptide Y [22] and proinsulin [23, 24]. We have used PC2 knock-out mice [27] to confirm the involvement of this enzyme in the natural processing of PE
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