Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group.
The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn+) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the Km value for agmatine (from 1.1 +/- 0.2 mM to 6.3 +/- 0.3 mM) and the Ki value for competitive inhibition by Gdn+ (from 15.0 +/- 0.1 mM to 44.2 +/- 2.1 mM), but the Ki value for putrescine inhibition (2.8 +/- 0.2 mM) was not altered. The tryptophan fluorescence properties (lambdamax = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 --> Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.
Carvajal
Nelson
N
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Chile. ncarvaja@udec.cl
Orellana
María S
MS
Salas
Mónica
M
Enríquez
Paula
P
Alarcón
Ricardo
R
Uribe
Elena
E
López
Vasthi
V
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D014644:Genetic Variation; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014512:Ureohydrolases
D005973:Glutamine; D014512:Ureohydrolases; C019625:agmatinase; D019791:Guanidine