Mutations of the PC2 Substrate Binding Pocket Alter Enzyme Specificity

Abstract

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser 206 , Thr 271 , Asp 278 , ArgGlu 282 , AlaSer 323 , Leu 341 , Asn 365 , and Ser 380 , which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser 206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.Prohormone convertase (PC) 1 2 belongs to the family of mammalian calcium-dependent serine proteases known as subtilisin/kexin-like proprotein convertases. The seven members of this family, furin, PC2, PC1/3, PACE4, PC4, PC5/6, and PC7/8, are the major endoproteolytic processing enzymes of the secretory pathway (1). PCs are multidomain proteins, all consisting of a prodomain, believed to assist in correct folding of the enzyme (2-4), a catalytic domain with some homology to bacterial subtilisin, and a P-or homo-B domain that plays an essential role in regulating stability and modulating activity (4). The proprotein convertases are thought to be responsible for the proteolytic maturation of precursors of a multitude of proteins and peptides, such as polypeptide hormones, neuropeptides, proteases, blood coagulation factors, adhesion factors, and receptors (reviewed in Ref. 5). PCs process these precursors at mono or dibasic residues. Furin, the best studied convertase, most frequently cleaves following the consensus sequence RX(R/K)R (5); PACE4 has a similar specificity (6). Other PCs also accept Lys in the P1 position (for nomenclature see Ref. 7) and are less dependent on the presence of basic residues in the P4 position (8).The prohormone convertases PC2 and PC1 are expressed mainly in neuroendocrine tissues and act in the regulated secretory pathway. These enzymes are involved in the proteolytic maturation of most if not all neuropeptide precursors, including the opioid peptide precursors proopiomelanocortin, proenkephalin, and prodynorphin (9). PC1 has been shown to be...

Neuropeptidomic analysis establishes a major role for prohormone convertase‐2 in neuropeptide biosynthesis

“…Those peptides that partially decreased in the PC2 KO mice but which were still detectable in the mutants presumably are cleaved by both PC2 and other endopeptidases, while those peptides showing a very large decrease in the PC2 KO mice represent cleavage products unique to PC2 that another enzyme cannot perform. Analysis of the cleavage sites of these three groups of peptides is generally consistent with previous studies testing purified PC2 with synthetic substrates (Smeekens et al 1992;Lamango et al 1996;Johanning et al 1998;Steiner 1998;Kacprzak et al 2005). In a comparison of tri-peptides with a flurogenic group in the P1¢ position, Arg-Arg and Lys-Arg sequences were found to be cleaved by PC2 with comparable kinetics (Johanning et al 1998).…”

“…Previous studies on purified enzyme also support the importance of Pro in the P1′ and P2′ positions in conferring exclusive or preferred processing of a site by PC2 and not the other PCs (Day et al. 1998; Kacprzak et al. 2005).…”

“…However, previous tendencies noted by Pan et al, such as the presence of a basic residue in the P3 position of preferred PC2 substrates, did not hold up when the larger number of peptides and brain regions was considered. Previous studies on purified enzyme also support the importance of Pro in the P1¢ and P2¢ positions in conferring exclusive or preferred processing of a site by PC2 and not the other PCs (Day et al 1998;Kacprzak et al 2005). However, other trends noted from the studies using purified PC2 and synthetic substrates, such as charged residues in the P2¢ position or positively charged residues in the P1¢ or P3¢ positions (Kacprzak et al 2005), do not appear to predict whether peptides require PC2 in vivo (Fig.…”

“…2005). However, other trends noted from the studies using purified PC2 and synthetic substrates, such as charged residues in the P2′ position or positively charged residues in the P1′ or P3′ positions (Kacprzak et al. 2005), do not appear to predict whether peptides require PC2 in vivo (Fig.…”

Is gene duplication a viable explanation for the origination of biological information and complexity?

“…A random mutation in the active site could well lead to an “advantageous” outcome in a particular environment owing to a shift in catalytic activity. However, the evidence suggests this would entail an alteration in the particular specificity pattern30. Therefore, it would mean that an increase of uncertainty and more erratic behavior, with respect to the overall and net effect(s), is a consequence of such a development.…”