Catalytic site studies on tuna (Thunnus albacares) pyloric caeca aminopeptidase.
Tuna pyloric caeca aminopeptidase (tAP) is a glycosylated zinc-metalloenzyme containing apparently two identical subunits. The enzyme is reversibly inhibited in a time-dependent manner by amastatin. Slow development of tAP inhibition by this inhibitor could be demonstrated. Dissociation of the complex of tAP with amastatin is also slow. Two molar equivalents of the inhibitor are bound by the enzyme suggesting the presence of one catalytic site in each subunit. Chemical modification of tAP with 1-cyclohexyl-3-(2-morpholinoethyl) carbonyl-metho-p-toluene sulfonate and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone revealed the presence of essential acidic amino acid residues probably located at the active site. Compatible with the presence of arginine and tyrosine residues at the catalytic site of most metalloproteinases, tAP is reversibly inhibited 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 amastatin 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.
Hajjou
M
M
Laboratoire de Biologie Marine, Concarneau, France.
Le Gal
Y
Y
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001120:Arginine / Q000032:analysis; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate; D010455:Peptides; D010658:Phenylglyoxal; D011804:Quinolines / Q000494:pharmacology; D013774:Tetranitromethane; D014413:Tuna / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D010455:Peptides; D011804:Quinolines; C074111:N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-4-toluolsulfonate; D003028:CME-Carbodiimide; C000470:EEDQ; C016977:amastatin; D001120:Arginine; D000626:Aminopeptidases; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal