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Jaffe EK , Abrams WR , Kaempfen HX , Harris Jr KA
5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc
Biochemistry. 1992 ;31(7) :2113-2123
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Abstract
Porphobilinogen synthase (PBGS) is a Zn(II) metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA). The nitrogen of the first substrate ends up in the pyrrole ring of product (P- side ALA); by contrast, the nitrogen of the second substrate molecule remains an amino group (A-side ALA). A reactive mimic of the substrate molecules, 5- chlorolevulinate (5-CLA), has been prepared and used as an active site directed irreversible inhibitor of PBGS. Native octameric PBGS binds eight substrate molecules and eight Zn(II) ions, with two types of sites for each ligand. As originally demonstrated by Seehra and Jordan [(1981) Eur. J. Biochem. 113, 435-446], 5-CLA inactivates the enzyme at the site where one of the two substrate molecules binds, and modification at four sites per octamer (one per active site) affords near-total inactivation. Here we report that 5- CLA-modified PBGS (5-CLA-PBGS) can bind up to four substrate molecules and four Zn(II) ions. Contrary to the conclusion of Seehra and Jordan, we find that the preferential site of 5-CLA inactivation is the A-side ALA binding site. On the basis of the dissociation constants, the metal ion binding sites lost upon 5-CLA modification are assigned to the four catalytic Zn(II) sites. 5-CLA-PBGS is shown to be modified at cysteine-223 on half of the subunits. We conclude that cysteine-223 is near the amino group of A-side ALA and propose that this cysteine is a ligand to the catalytic Zn(II). The vacant substrate binding site on 5-CLA-PBGS is that of P-side ALA. We have used 13C and 15N NMR to view [4-13C]ALA and [15N]ALA bound to 5-CLA-PBGS. The NMR results are nearly identical to those obtained previously for the enzyme-bound P-side Schiff base intermediate [Jaffe et al. (1990) Biochemistry 29, 8345-8350]. It appears that, in the absence of the catalytic Zn(II), 5-CLA-PBGS does not catalyze the condensation of the amino group of the P-side Schiff base intermediate with the C4 carbonyl derived from 5-CLA. On this basis we propose that Zn(II) plays an essential role in formation of the first bond between the two substrate molecules.
Notes
00062960 (ISSN) Cited By: 19; Export Date: 31 May 2006; Source: Scopus CODEN: BICHA Language of Original Document: English Correspondence Address: Jaffe, E.K.; Fox Chase Cancer Center; Institute for Cancer Research; 7701 Burholme Ave. Philadelphia, PA 19111, United States Chemicals/CAS: aminolevulinic acid, 106-60-5; porphobilinogen synthase, 9036-37-7; zinc, 7440-66-6, 5-chloro-4-oxopentanoic acid, 60254-71-9; Levulinic Acids; Porphobilinogen Synthase, EC 4.2.1.24; Somatostatin, 51110-01-1; Zinc, 7440-66-6