Structural and functional roles of asparagine 175 in the cysteine protease papain.
The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175-->Gln and Asn175-->Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.
Vernet
T
T
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Tessier
D C
DC
Chatellier
J
J
Plouffe
C
C
Lee
T S
TS
Thomas
D Y
DY
Storer
A C
AC
Ménard
R
R
D000595:Amino Acid Sequence; D001216:Asparagine; D001665:Binding Sites; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000737:chemistry* / Q000502:physiology; D017510:Protein Folding; D013329:Structure-Activity Relationship
D001216:Asparagine; D010206:Papain