Dissection of the early steps in the porphobilinogen synthase catalyzed reaction. Requirements for Schiff's base formation.
The porphobilinogen (PBG) synthase catalyzed reaction requires both Zn(II) and reducing equivalents for the production of PBG from two molecules of 5-aminolevulinic acid (ALA). An early step in the reaction is the production of a Schiff's base between PBG synthase and one ALA molecule. Because both substrate molecules are chemically identical, there had been no evidence of enzyme-catalyzed partial reactions of ALA under conditions where PBG is not formed. In this study, NaBH4 was used to trap the Schiff's base formed between substrate ALA and active holo-PBG synthase, inactive apo-PBG synthase, and inactive methylmethanethiosulfonate-modified apo-PBG synthase. ALA-dependent NaBH4 inactivation of these enzyme forms was quantified at 50-62, 94-97, and 93-96% inactivation, respectively. [4-14C]ALA was used to determine the stoichiometry of Schiff's base trapping which was 2.3, 3.5-4.0, and 3.4 per octamer for holoenzyme, apoenzyme, and methylmethanethiosulfonate-modified apoenzyme, respectively. These results are consistent with four active sites per octamer or half-of-the-sites reactivity. We conclude that the production of the Schiff's base formed between one ALA molecule and the enzyme requires neither Zn(II) nor reduced enzyme sulfhydryl groups. Furthermore, the possible number of kinetic schemes for formation of the quaternary complex of enzyme, Zn(II), and two ALA moieties, one as the Schiff's base, has been reduced from 12 to 3. This is the first demonstration of a partial reaction catalyzed by PBG synthase with the natural substrate ALA under conditions which do not support PBG formation. Thus, we have opened the way toward investigating the partial reactions which may precede Zn(II) participation in the PBG synthase reaction.
D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D011162:Porphobilinogen / Q000096:biosynthesis; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012545:Schiff Bases; D015032:Zinc / Q000494:pharmacology
D001051:Apoenzymes; D001894:Borohydrides; D046911:Macromolecular Substances; D012545:Schiff Bases; D011162:Porphobilinogen; C025364:sodium borohydride; D000622:Aminolevulinic Acid; D008741:Methyl Methanesulfonate; D000623:Porphobilinogen Synthase; D015032:Zinc