An isozyme-selective affinity label for rat hepatic acetyltransferases.

Area of Science:

• Biochemistry
• Enzymology
• Pharmacology

Background:

• N-acetyltransferase (NAT) enzymes exist in multiple forms with distinct catalytic activities.
• Understanding the specific roles and structures of NAT isozymes is crucial for drug development and toxicology.

Purpose of the Study:

• To differentiate and characterize the activities of N-acetyltransferase I (NAT I) and N-acetyltransferase II (NAT II) isozymes.
• To identify and investigate selective inhibitors for NAT isozymes, particularly NAT II.
• To explore the utility of selective inhibitors as probes for enzyme active site studies.

Main Methods:

• Affinity chromatography was used to separate NAT isozymes from rat hepatic cytosol.
• Enzyme kinetics and inhibition studies were performed using specific substrates and potential inhibitors.
• 1-(Fluoren-2-yl)-2-propen-1-one (vinyl fluorenyl ketone, VFK) was tested for its inhibitory effects on NAT I and NAT II.

Main Results:

• Two distinct NAT fractions, NAT I and NAT II, were separated based on their catalytic activities.
• Vinyl fluorenyl ketone (VFK) selectively and irreversibly inactivated NAT II at concentrations 200-fold lower than required for NAT I inactivation.
• Substrates and products of NAT II protected the enzyme from VFK inactivation, confirming VFK as an active site-directed inhibitor.

Conclusions:

• Vinyl fluorenyl ketone (VFK) is a potent and selective affinity label for N-acetyltransferase II (NAT II).
• The conjugated ketone group in VFK is essential for its enzyme inactivation activity.
• VFK and similar agents can serve as valuable tools for investigating acetyltransferase multiplicity and active site characteristics.