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 presenc e of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector c omplex (12 mM) was essentially equal to the K-i value for this compound act ing as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His 163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-t ype activity, without any change in K-m for agmatine or K-i for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivat ions. We conclude that His163 plays an important role in the catalytic func tion of agmatinase, but it is not directly involved in substrate binding. ( C) 1999 Academic Press.