Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling.
Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was reduced to about 5% of wild-type activity. Tryptophan emission fluorescence (lambdamax = 340 nm), and CD spectra were nearly identical for wild-type and D153N agmatinases. The Km value for agmatine (1.6 +/- 0.1 mm), as well as the Ki for putrescine inhibition (12 +/- 2 mm) and the interaction of the enzyme with the required metal ion, were also not altered by mutation. Three-dimensional models, generated by homology modelling techniques, indicated that the side chains of Asp153 and Asn153 can perfectly fit in essentially the same position in the active site of E. coli agmatinase. Asp153 is suggested to be involved, by hydrogen bond formation, in the stabilization and orientation of a metal-bound hydroxide for optimal attack on the guanidinium carbon of agmatine. Thus, the disruption of this hydrogen bond is the likely cause of the greately decreased catalytic efficiency of the D153N variant.
Salas
Mónica
M
Departamento de Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
Rodríguez
Rolando
R
López
Nelia
N
Uribe
Elena
E
López
Vasthi
V
Carvajal
Nelson
N
D000595:Amino Acid Sequence; D000818:Animals; D001119:Arginase / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D002942:Circular Dichroism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014512:Ureohydrolases / Q000737:chemistry*
D001224:Aspartic Acid; D008345:Manganese; D004492:Edetic Acid; D014512:Ureohydrolases; D001119:Arginase; C019625:agmatinase