Abstract

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 (?max = 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.