CONTRIBUTION OF AROMATIC MOIETIES OF TYROSINE-133 AND OF THE ANIONIC SUBSITE TRYPTOPHAN-86 TO CATALYTIC EFFICIENCY AND ALLOSTERIC MODULATION OF ACETYLCHOLINESTERASE

Substitution of Trp-86, in the active center of human acetylcholineste rase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrat e and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, r eplacement of Glu-202, the only acidic residue proximal to the catalyt ic site, by glutamine resulted in a nonselective decrease in reactivit y toward charged and noncharged substrates or inhibitors. Thus, the qu aternary nitrogen groups of substrates and other active center ligands , are stabilized by cation-aromatic interaction with Trp-86 rather tha n by ionic interactions, while noncharged ligands appear to bind to di stinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutat ed enzymes suggests that the highly conserved Tyr-133 plays a dual rol e in the active center: (a) its hydroxyl appears to maintain the funct ional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp -86. In the absence of aromatic interactions between Tyr-133 and Trp-8 6, the tryptophan acquires a conformation that obstructs the active si te leading, in the Y133A enzyme, to several hundredfold decrease in ra tes of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylc holinesterase activity, induced by binding to the peripheral anionic s ites, proceeds through such conformational change of Trp-86 from a fun ctional anionic subsite state to one that restricts access of substrat es to the active center.