Abstract
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 enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.
Highlights
Introduction of Mutations into theE. coli Expression Vector PIN III-Expression of AdoMetDC Mutant cDNAs in Reticulocyte Lysates-RNA was prepared by transcription of the original andA3-Wild type AdoMetDC wassubcloned into the PIN III-A3 mutant AdoMetDC sequences in the pGEM3Z(f-)vector using vector containing the stronger promotpe-r5 [20]as described previously T7 RNA polymerase
Western blot analysis a reticulocyte lysate system in which we have shown previously that of proteins reactive withanti-AdoMetDC polyclonal antibodies [12] was processing occurs andis stimulated by putrescine [16].I n vitro trans- performed by the ECL method(AmershamCorp.)accordingtothe lation reactions to generate the proenzyme werfeorr3u0n minat 30 "C manufacturer's instructions. without added putrescine, thecnycloheximide and RNase A were added Miscellaneous Methods-Protein wasmeasured by the method of (100 PMand 15pg/150pl invitro translation reaction, respectively), and Bradford
It isclear that the humaAndoMetDC and mutant cDNAs were well expressed in the bacteria and that processing occurred in all mutants testedexcept the S68A mutant, which cannot form pyruvate at the amino terminuosf the a subunit [8].Based on densitometric scanning of Western blots, theamounts of processed AdoMetDC did not differ greatly between the wild type and the mutant(sTable 11).there was a larger amountof the proenzyme present inall of the cells expressing the mutant cDNAs so that 26-50%of these mutanAt doMetDC proteins werepresent in theiurnprocessed form, as compared with 7% for the wild type enzyme
Summary
A t appropriatetimepoints, mixed with pl of gel loading buffer, heated 10 min in boiling water, and either loaded onto 1.5-mm 12.5%. For determinations of the effect of putrescine on catalytic activity, in son which is shown in Fig. 1; this showed lack of vitro translation reactions (90 pl) were run in the presenceof 0.2 m~ conservation at the already tested Glu-135 and Glu-183 resiputrescine (to achieve maximal proenzyme processing) for 90 min at 30 "C, eluted through a Sephadex G-25 spin column (Select-D, from 5'-3', West Chester, PA) to remove putrescine from the lysates. Two such in vitro translation reactions were combined after elution, mixed, and split inAtodoMetDC activity assays containing200 pM AdoMet with or without 2 m~ putrescine. Activity assayswere performed on 50- or 75-pl invitro translationreactionsasdetailed under "ExDerimental Procedures" usin-g 0.2mM AdoMet as substrate
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