CATALYTIC SITE STUDIES ON TUNA (THUNNUS ALBACARES) PYLORIC CECA AMINOPEPTIDASE

Tuna pyloric caeca aminopeptidase (tAP) is glycosylated zinc-metalloen zyme containing apparently two identical subunits. The enzyme is rever sibly inhibited in a time-dependent manner by amastatin. Slow developm ent of tAP inhibition by this inhibitor could be demonstrated. Dissoci ation of the complex of tAP with amastatin is also slow, Two molar equ ivalents of the inhibitor are bound by the enzyme suggesting the prese nce of one catalytic site in each subunit. Chemical modification of tA P with exyl-3-(2-morpholinoethyl)carbonyl-metho-p-toluene sulfonate an d N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone revealed the presence o f essential acidic amino acid residues probably located at the active site, Compatible with the presence of arginine and tyrosine residues a t the catalytic site of most metalloproteinases, tAP is reversibly inh ibited by phenylglyoxal and inactivated by tetranitromethane in a time -dependent fashion, The rate of inhibition by these modifiers could be significantly decreased if the enzyme was previously treated with ama statin suggesting that the modified amino acid residues are located at the catalytic site, Diethylpyrocarbonate did not affect the activity of both native and zinc-depleted tAP suggesting that histidine is not involved in the zinc-ligand formation.