EXPRESSION OF MAMMALIAN S-ADENOSYLMETHIONINE DECARBOXYLASE IN ESCHERICHIA-COLI - DETERMINATION OF SITES FOR PUTRESCINE ACTIVATION OF ACTIVITY AND PROCESSING

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature e nzyme activity. To determine sites of putrescine interaction with AdoM etDC, putrescine stimulation of both proenzyme processing and catalyti c activity was tested with mutant AdoMetDCs in which specific amino ac id residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and t he previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putr escine without an effect on the processing rate in the absence of putr escine. Mutations E11K, as well as Y112A and L259Stop, completely abol ished processing regardless of putrescine concentration, whereas mutat ion E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proe nzyme processing. The effects of mutations on enzyme activity were det ermined using AdoMetDC protein produced in Escherichia coli and purifi ed by affinity chromatography. Mutation E11Q completely inactivated th e enzyme, mutation E133Q reduced the catalytic constant by >10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimul ate the activity of mutants E178Q and E256Q but did activate mutants E 133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu -256 are critical residues in the putrescine stimulation of AdoMetDC p roenzyme processing and that Glu-178 and Glu 256 are critical for putr escine stimulation of AdoMetDC catalytic activity.