pH studies to elucidate the chemical mechanism of penicillin acylase from Kluyvera citrophila.
The variation with pH of the kinetic parameters of penicillin acylase from Kluyvera citrophila has been used to gain information about the chemical mechanism of the reaction catalysed by the enzyme. The pH-dependence of log (V/Km) for penicillin G showed that a group with a pK value over 4.7 must be deprotonated and that a group with a pK value over 9.7 must be protonated in the free enzyme for activity. The solvent perturbation and temperature studies indicated that these groups are respectively of cationic and neutral acid type with ionization enthalpies of 29.7 and 111 kJ/mol. It was proved that penicillin G sulphoxide is a reversible linear competitive inhibitor with respect to the hydrolysis of penicillin G. The similarity of the pH profile and the magnitude of the pK values derived from the dissociation constant, Ki, suggest that both groups are concerned with the binding of penicillin G and its analogues to the enzyme. It is proposed that binding of substrate involves the formation of hydrogen bonds between the substrate and the essential ionizable groups in the enzyme which lie within the hydrophobic environment of the active site of penicillin acylase. This suggestion is supported by the finding that the profile of V (Vmax.) is similar to the V/Km profile, except that the low and high pK values are respectively shifted downward and upward due to the entry of substrate. Moreover, the bell shape of the V profile indicated that they are also essential in the catalytic steps subsequent to binding.
Martín
J
J
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain.
Prieto
I
I
Mancheño
J M
JM
Barbero
J L
JL
Arche
R
R
D002384:Catalysis; D004755:Enterobacteriaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010400:Penicillin G / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
C059126:penicillin G sulfoxide; D010399:Penicillin Amidase; D010400:Penicillin G