Porphobilinogen synthase modification with methylmethanethiosulfonate. A protocol for the investigation of metalloproteins.
A stable, reversibly sulfhydryl-modified, Zn2+-free porphobilinogen synthase (mod-apo-PBG synthase) has been prepared using methylmethanethiosulfonate. Mod-apo-PBG synthase prepared from holo-PBG synthase using [methyl-14C]methanethiosulfonate incorporated three thiomethyl groups/subunit. When apo-PBG synthase was prepared using EDTA alone, subsequent reaction with [methyl-14C]methanethiosulfonate resulted in incorporation of only two thiomethyl groups/subunit. Mod-apo-PBG synthase was catalytically inactive and contained less than 0.1 mol of Zn/mol of octameric enzyme; it could be reconstituted to full activity using 2-mercaptoethanol and Zn2+. A variety of metal ions were screened for their ability to reconstitute and/or reactivate mod-apo-PBG synthase. Only Zn2+ and Cd2+ reconstitute mod-apo-PBG synthase to full activity. When comparing mod-apo-PBG synthase prepared from holo-PBG synthase in the presence of EDTA with mod-apo-PBG synthase prepared from holo-PBG synthase in the absence of EDTA, no difference was detected in either Zn content, stoichiometry of 14C-labeling, or kinetic behavior. We have confirmed both the observations that four Zn2+/mol of octameric apoenzyme are necessary for full catalytic activity and that holoenzyme, isolated in the presence of 10 microM ZnCl2, contains eight Zn2+/octamer. The additional four binding sites are not catalytically important. Methylmethanethiosulfonate modification is presented as a generally useful method for the investigation of metalloproteins because it provides a route for the preparation of stable apoproteins and a direct method for metal ion replacement.
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002417:Cattle; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000623:Porphobilinogen Synthase / Q000378:metabolism*; D011485:Protein Binding; D015032:Zinc / Q000494:pharmacology
D001059:Apoproteins; D008024:Ligands; D046911:Macromolecular Substances; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D000623:Porphobilinogen Synthase; D015032:Zinc