THERMODYNAMIC PROFILES OF PENICILLIN-G HYDROLYSIS CATALYZED BY WILD-TYPE AND MET-]ALA168 MUTANT PENICILLIN ACYLASES FROM KLUYVERA-CITROPHILA

被引:24
作者
MARTIN, J
PRIETO, I
BARBERO, JL
PEREZGIL, J
MANCHENO, JM
ARCHE, R
机构
[1] UNIV COMPLUTENSE MADRID, FAC QUIM, DEPT BIOQUIM & BIOL MOLEC 1, E-28040 MADRID, SPAIN
[2] ANTIBIOT FARMA SA, DEPT GENET MOLEC, MADRID, SPAIN
关键词
Penicillin acylase; Reaction thermodynamics; Site directed mutagenesis;
D O I
10.1016/0167-4838(90)90158-C
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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≠) 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. © 1990.
引用
收藏
页码:133 / 139
页数:7
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