New insights on the specificity of penicillin acylase.
In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.
Roa
A
A
Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
Castillón
M P
MP
Goble
M L
ML
Virden
R
R
García
J L
JL
D004952:Esters; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D009596:Nitrophenols; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010400:Penicillin G / Q000031:analogs & derivatives / Q000494:pharmacology; D010648:Phenylacetates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004952:Esters; D009596:Nitrophenols; D010648:Phenylacetates; D011994:Recombinant Proteins; D010399:Penicillin Amidase; C025136:phenylacetic acid; D010400:Penicillin G