Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila.
The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.
Martín
J
J
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain.
Prieto
I
I
Barbero
J L
JL
Pérez-Gil
J
J
Mancheño
J M
JM
Arche
R
R
D000409:Alanine; D000581:Amidohydrolases / Q000378:metabolism*; D001665:Binding Sites; D004755:Enterobacteriaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008715:Methionine; D009154:Mutation; D010399:Penicillin Amidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity; D013816:Thermodynamics
D008715:Methionine; D000581:Amidohydrolases; D010399:Penicillin Amidase; D000409:Alanine; D010400:Penicillin G