Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation

Abstract

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, β-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver–Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K m increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and β-ketoacyl reduction reaction, and acetylacetate, the substrate of the β-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3–4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.