Mutations of the PC2 substrate binding pocket alter enzyme specificity

Mutations of the PC2 substrate binding pocket alter enzyme specificity

Author Kacprzak, M. M. Google Scholar
Than, M. E. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Bode, W. Google Scholar
Lindberg, I Google Scholar
Institution Louisiana State Univ
Max Planck Inst Biochem
Universidade Federal de São Paulo (UNIFESP)
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 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.
Language English
Date 2005-09-09
Published in Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 280, n. 36, p. 31850-31858, 2005.
ISSN 0021-9258 (Sherpa/Romeo, impact factor)
Publisher Amer Soc Biochemistry Molecular Biology Inc
Extent 31850-31858
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000231665200064

Show full item record


File Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)




My Account