Effect of chaotropic agents on the structure-function of recombinant acylpeptide hydrolase.
Acylpeptide hydrolase, a new class the serine-type peptidase, belongs to the alpha,beta hydrolase group of proteins. The tetrameric enzyme showed varying degree of stability in the presence of 1-8 M urea. The enzyme displayed about 15% of its original activity when treated with 8 M urea for 1 h at 25 degrees C. Complete recovery of the enzyme activity was observed on dialysis or dilution (50-fold) of the denatured enzyme. However, complete abolition of the enzyme activity was observed in the presence of 1 M GnHCl. Dialysis of the 1 M GnHCl-treated enzyme resulted in 15-20% recovery of the enzyme activity. The fluorescence emission spectra of the native enzyme at 337 nm showed a red shift up to 16 nm in 8 M urea and 18 nm in the presence of 4 M GnHCl. Native enzyme during far-UV circular dichroism spectroscopy exhibited predominantly beta-sheet structure. The enzyme lost its secondary structure at urea concentrations of 2 M and higher, whereas the tertiary structure was minimally perturbed below 4 M urea. However, in 1 M GnHCl the enzyme lost both its secondary and tertiary structures and the enzyme was found to dissociate into monomers of 70 kDa. Both monomeric and dimeric species were observed after 24-h dialysis of the enzyme denatured with GnHCl indicating the reassociation process. Both monomer and dimers forms recovered after dialysis were active.
Department of Ophthalmology, University of Missouri, Columbia 65212, USA.
R
R
Senthilkumar
K Krishna
KK
Sharma
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013552:Swine; D014508:Urea / Q000494:pharmacology*
D011994:Recombinant Proteins; D014508:Urea; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase; D019791:Guanidine