Contribution of the carbohydrate moiety to conformational stability of thecarboxypeptidase Y - High pressure study

The process of pressure-induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low tempera ture 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 co nformational changes characterized by a slight decrease in the center of th e spectral mass of intrinsic fluorescence, whereas no changes in 8-anilino- 1-naphthalene sulfonic acid binding fluorescence were observed and 80% of t he catalytic activity remained. Higher pressure (150-500 MPa) induced furth er conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8-a nilino-1-naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of m olten globule-like state. A further increase, in pressure (above 550 MPa) i nduced transition from this first molten globule-like state to a second mol ten globule-like state. This two-stage denaturation process can be explaine d by assuming the existence of two independent structural domains in the ca rboxypeptidase molecule. A similar three-transition process was found for unglycosylated carboxypept idase Y,but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate tha t the carbohydrate moiety protects carboxypeptidase Y against pressure-indu ced denaturation. The origin of the protective effects is discussed based o n the known crystallographic structure of CPY.