Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase.
The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.
Kashem
M A
MA
Department of Chemistry, Cornell University, Ithaca, NY 14853.
Hammes
G G
GG
D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008099:Liver / Q000201:enzymology*; D009994:Osmolar Concentration; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D013696:Temperature
D003432:Cross-Linking Reagents; D064429:Fatty Acid Synthases