Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2.
The high-resolution crystal structure of kexin (Kex2) in complex with a peptidyl-chloromethylketone inhibitor containing a noncognate lysine at the P(1) position provides the structural basis for the differential lysine/arginine selectivity that defines the prohormone (proprotein) convertase (PC) family. By comparison with the previous structures of Kex2 and furin, this structure of the acylated enzyme provides a basis for the observed decrease in the acylation rate with substrates containing a lysine at P(1) and the absence of an effect on the deacylation rate without involving mobility of the S(1) lid. The structure of the complex shows that a secondary subsite in the S(1) pocket is present, and that this site recognizes and binds the P(1) lysine in a more shallow fashion than arginine. This results in a displacement of the bound peptide away from the S385 nucleophile relative to substrates containing a P(1) arginine. It is concluded that this alternate binding site and resultant displacement of the scissile bond in the active site results in the observed decrease in the acylation rate. Studies of the inactivation kinetics of Kex2 by two peptidyl chloromethylketone inhibitors demonstrates that the selectivity between lysine and arginine at the P(1) position arises at the acylation step, consistent with what was observed with peptidyl substrates [Rockwell NC, Fuller RS (2001) J Biol Chem 276:38394-38399].
Wheatley
Joshua L
JL
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Holyoak
Todd
T
D001120:Arginine / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008239:Lysine / Q000378:metabolism*; D043484:Proprotein Convertases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae; D008239:Lysine