Negative functional interference between two active centers is indicated in animal fatty acid synthetase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Fatty acid synthetase (FAS) is a multi-enzyme complex crucial for fatty acid biosynthesis.
• The Harderian gland in guinea-pigs is a model for studying FAS regulation.
• Understanding enzyme kinetics and active site interactions is vital for metabolic research.

Purpose of the Study:

• To investigate the effect of phenylmethanesulfonyl fluoride (PMSF) on guinea-pig Harderian gland fatty acid synthetase activity.
• To elucidate the relationship between thioesterase activity inhibition and overall enzyme function.
• To explore the interaction dynamics between the active centers of the native FAS enzyme.

Main Methods:

• Enzyme inhibition assays using varying concentrations of phenylmethanesulfonyl fluoride.
• Spectrophotometric determination of fatty acid synthetase and thioesterase activities.
• Analysis of enzyme kinetics in native and heterodimeric forms.

Main Results:

• Overall fatty acid synthetase activity decreased quadratically with increasing inhibition of thioesterase activity.
• The primary inhibition target was identified as the thioesterase activity.
• Heterodimers formed between native and thioesterase-less monomers exhibited higher specific activity than the native enzyme.

Conclusions:

• The results indicate a negative interference between the two active centers of the native fatty acid synthetase.
• Thioesterase activity plays a critical role in the overall catalytic efficiency of the enzyme.
• Enzyme structure-activity relationships in FAS can be modulated by specific inhibitors.