Arylamine N-Acetyltransferase 1 Brendan Thompson

Background Nicotine, Cotinine, Chlorphenamine What do they all have in common? Arylamine drugs and toxins metabolized by arylamine N-acetyltransferase 1 (NAT-1) Arylamine N-acetyltransferase 1 is a metabolic enzyme that is used in both bacteria and eukaryotes to metabolize foreign chemicals such as the ones shown on this slide. These chemicals are nicotine and cotinine, both found in cigarettes and cigarette smoke, and chlorphenamine is an antihistamine sold routinely. Each structure contains an arylamine, a heterocyclic, aromatic structure that is specific to the active site of NAT-1. The enzyme functions with acetyl-CoA to ultimately transfer its acetyl group onto the arylamine, preventing the potential harmful oxidant from damaging DNA and potentially initiating cancer growth. The source of the enzyme’s function comes from a catalytic triad consisting of Cys68-His107-Asp122. Background

Hydropathy Plot

Hydrophobic Interior containing the conserved catalytic triad Cys68 is critical for enzyme structure and function, demonstrated in several mutagenesis studies. NAT-1 is a cytosolic protein that has a primarily hydrophobic core that holds the catalytic triad. Why it is mostly hydrophobic? The last step of the triad mechanism is the hydrolysis of the acetyl-arylamine. The hydrophobic interior of the protein allows for an extremely slow rate of hydrolysis, which is critical for proper function of the protein. If it was hydrophilic, then water would come in too quickly, hydrolyzing the acetyl-sulfhydryl intermediate which you can see in the right hand picture, not allowing for acetylation of the arylamine. Also talk about the C68Y mutation study. Structure

Sequence Alignment    This catalytic triad is conserved in all organisms. This can be seen from the sequence alignment shown here, which compares human, rat, mouse, bacterial, rabbit, and six other diverse organisms. Even though the organisms vary greatly, each one of them has these three essential amino acids within their active site. This is proof that the triad is critical for protein function.   Sequence Alignment

Mechanism Acetyl Transfer to Cys68 Residue Transfer to Arylamine and Hydrolysis Mechanism

Structure Green: Analog of CoA’s Pantotheine arm Blue: Hydrophobic Amino Acids and Van der Waals Interactions (F37, F93, L209, F217, L288) Purple: Side Chain H-bonding to the PPi (Y208, T214) Orange: Backbone H-bonding to the PPi (T103, G104) Acetyl CoA binding to Phe37, Phe93, Leu209, Phe217, Leu288 participating in Van der Waals interactions on the pantotheine arm of CoA, while the pyrophosphate group of CoA participates in hydrogen bonding with amine nitrogens on Thr103 and Gly104, and with the hydroxyl groups of Tyr208 and Thr214. Possibly creates a cavity for entry of water for hydrolysis when it leaves. Structure

Structure

Connections to Cancer? N-acetylation vs. O-acetylation Many studies suggest links between NAT-1 and its isozyme NAT-2 and several different cancers, especially cancers heavily related to cigarette smoke. Can anybody guess why? Potentially because cigarette smoke contains nicotine and cotinine, two foreign chemicals that are arylamines. When NAT-1 and NAT-2 reach their Vmax, they cannot turn over any faster, leaving the potential for oxidative damage to DNA. Another issue is a separate mechanism that is possible. Instead of N-acetylation, when saturated, the enzymes can actually O-acetylate. Where N-acetylation is associated with detoxification and metabolism of foreign chemicals, O-acetylation is associated with bioactivation into carginogens. N-acetylation vs. O-acetylation Connections to Cancer?