A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding.
The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.
Moparthi
Satish Babu
SB
Department of Chemistry, Linköping University, Sweden.
Hammarström
Per
P
Carlsson
Uno
U
D001120:Arginine / Q000502:physiology*; D024402:Carbonic Anhydrase II / Q000378:metabolism; D020134:Catalytic Domain; D021984:Cyclophilin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding
D010455:Peptides; D016572:Cyclosporine; D001120:Arginine; D024402:Carbonic Anhydrase II; D021984:Cyclophilin A