M. Hajjou et Y. Legal, CATALYTIC SITE STUDIES ON TUNA (THUNNUS ALBACARES) PYLORIC CECA AMINOPEPTIDASE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1251(2), 1995, pp. 139-144
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.