[Intracellular aminopeptidase from Xanthomonas rubrilineans, hydrolyzing alpha-amino acid esters and cefalexin].
The aminopeptidase was isolated from cell-free extracts of Xanthomonas rubrilineans by protein precipitation by isopropyl ester with subsequent purification by affinity chromatography on CABS-Sepharose, bacitracin-Sepharose, gel filtration through Sephadex G-200 and ultrafiltration, the total yield being 32% with 2200-fold purification. The enzyme was homogeneous during SDS-PAAG electrophoresis. Apart from the broad spectrum of the peptidase activity, aminopeptidase possesses a hydrolase activity towards beta-lactam antibiotics and an esterase activity towards L- and D-amino acids. Besides, this enzyme catalyzes the acetyl transfer reaction during cephalexin synthesis from the D-phenylglycine ester and 7-aminodesacetoxycephalosporanic acid. The maximal enzyme activity during L-Ala-pNA and cephalexin hydrolysis is manifested at pH 6.5. The enzyme is stable at pH 4.0-8.0 and is inhibited by o-phenanthroline, p-chloromercuribenzoate, hydrogen acetate and N-bromosuccinimide. The molecular mass of the enzyme is 270-280 kDa. The enzyme is a tetramer; the molecular mass of each of its four subunits is 70 +/- 2 kDa. The isoelectric point for the enzyme is 6.8. The amino acid composition of the enzyme appears as follows: Asp63, Thr33, Ser32, Glu72, Gly55, 1/2Cys3-4, Val45, Ile24, Leu53, Tyr23, Phe24, Lys23, His16, Arg36, Pro60, Met25, Ala55.
Krest'ianova
I N
IN
Uvarov
N N
NN
Rudenskaia
G N
GN
Tsibanov
V V
VV
Vasil'eva
L I
LI
Stepanov
V M
VM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D002506:Cephalexin / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D014974:Xanthomonas / Q000201:enzymology*
D000596:Amino Acids; D002413:Cations, Divalent; D004952:Esters; D008670:Metals; D000626:Aminopeptidases; D002506:Cephalexin
Vnutrikletochnaia aminopeptidaza iz Xanthomonas rubrilineans, gidrolizuiushchaia éfiry alpha-aminokislot i tsefaleksin.