Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y

Abstract

The process of pressure-induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8-anilino-1-naphthalene sulfonic acid binding fluorescences.Pressure-induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8-anilino-1-naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150±500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8-anilino-1-naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule-like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule-like state to a second molten globule-like state. This two-stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule.A similar three-transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure-induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY. . Although much is known about the structure and biosynthesis of the oligosaccharides in CPY, the definitive role of the associated sugar moiety in the structure and function of this enzyme has yet to be established. Studies carried out with carbohydrate-free CPY, obtained by growing yeast in the presence of an inhibitor of N-linked glycosylation or by mutation, showed that the carbohydrate moiety does not affect the processing of the CPY precursor, its accurate vacuolar targeting, or its transport, but the rate of transport of unglycosylated CPY through secretory pathway to the vacuole was reduced [5,8]. Complete removal of carbohydrate by endo b-N-acetylglucosaminidase H increased the respective rates of SDS-induced inactivation and proteolytic degradation by two and threefold, compared to the rates for glycosylated CPY [9]. However, neither the in-vivo nor the in-vitro stability and activity of unglycosylated CPY obtained by mutation were significantly different than that of wild-type CPY (WtCPY) [8]. KeywordsHigh pressure is increasingly used as a`reagentless denaturant' to study the protein denaturation [10]. This tool is especially well adapted to the study of cold denaturation of protein because pressure decreases the freezing point of water [...

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