Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase.
Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.
Carvajal
N
N
Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile. ncarvaja@udec.cl
Olate
J
J
Salas
M
M
López
V
V
Cerpa
J
J
Herrera
P
P
Uribe
E
E
D002384:Catalysis; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012395:Rose Bengal / Q000378:metabolism; D013379:Substrate Specificity; D014512:Ureohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012395:Rose Bengal; D006639:Histidine; D014512:Ureohydrolases; C019625:agmatinase; D004047:Diethyl Pyrocarbonate