The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.
The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.
Kato
Michiko
M
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan. mk@kais.kyoto-u.ac.jp
Sato
Yasuhiro
Y
Shirai
Kumiko
K
Hayashi
Rikimaru
R
Balny
Claude
C
Lange
Reinhard
R
D002152:Calorimetry, Differential Scanning; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000494:pharmacology; D043402:Cathepsin A / Q000737:chemistry*; D006031:Glycosylation; D006358:Hot Temperature; D008958:Models, Molecular; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D017433:Protein Structure, Secondary / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D002241:Carbohydrates; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D043402:Cathepsin A