The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding.
Antichymotrypsin, a member of the serpin superfamily, has been shown to form inactive polymers in vivo, leading to chronic obstructive pulmonary disease. At present, however, the molecular determinants underlying the polymerization transition are unclear. Within a serpin, the breach position is implicated in conformational change, as it is the first point of contact for the reactive center loop and the body of the molecule. W194, situated within the breach, represents one of the most highly conserved residues within the serpin architecture. Using a range of equilibrium and kinetic experiments, the contribution of W194 to proteinase inhibition, stability and polymerization was studied for antichymotrypsin. Replacement of W194 with phenylalanine resulted in a fully active inhibitor that was destabilized relative to the wild-type protein. The aggregation kinetics were significantly altered; wild-type antichymotrypsin exhibits a lag phase followed by chain elongation. The loss of W194 almost entirely removed the lag phase and accelerated the elongation phase. On the basis of our data, we propose that one of the main roles of W194 in antichymotrypsin is in preventing polymerization.
Pearce
Mary C
MC
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Cabrita
Lisa D
LD
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Ellisdon
Andrew M
AM
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Bottomley
Stephen P
SP
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D040681:Structural Homology, Protein; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000515:alpha 1-Antitrypsin; D014364:Tryptophan; D002918:Chymotrypsin