Mutations of the PC2 substrate binding pocket alter enzyme specificity.
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.
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Magdalena M
MM
Kacprzak
Manuel E
ME
Than
Luiz
L
Juliano
Maria A
MA
Juliano
Wolfram
W
Bode
Iris
I
Lindberg
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D045683:Furin / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D045664:Proprotein Convertase 1 / Q000235:genetics / Q000378:metabolism; D045706:Proprotein Convertase 2 / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity / Q000235:genetics
D045683:Furin; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D002118:Calcium