Mutations of the PC2 Substrate Binding Pocket Alter Enzyme Specificity

Magdalena M Kacprzak,Manuel E Than,Luiz Juliano,Maria A Juliano,Wolfram Bode,Iris Lindberg

doi:10.1074/jbc.m505567200

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 Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, 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 Ser206 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.

Highlights

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 Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin
Analysis of the PC2 Substrate Binding Pocket—PC2 and furin possess 37% identical amino acids; by taking only the catalytic domain into consideration, this homology rises to 55%
We focused our interest on residues that (i) differ at least between PC2 and PC1/furin and (ii) are PC2-specific, as defined by conservation among all PC2s, both invertebrate and vertebrate

Summary

Introduction

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 Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. 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]. 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]. To provide information on molecular contributions to enzyme specificity, we have constructed and analyzed mutant forms of PC2 which, based on the furin crystal structure [12] and computational modeling studies [13], contain PC1- or furin-specific residues rather than PC2-specific residues positioned in or near the substrate binding pocket

Objectives

Results

Conclusion