5-Chloro[1,4-13C]levulinic acid modification of mammalian and bacterial porphobilinogen synthase suggests an active site containing two Zn(II).
5-Chloro[1,4-13C]levulinic acid ([1,4-13C]CLA) is an active site-directed inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrically condenses two molecules of 5-aminolevulinic acid (ALA) which are called A-side ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. [1,4-13C]CLA modifies bovine PBGS at the A-side ALA binding site. The C4 chemical shift indicates an intact keto moiety; the C1 chemical shift indicates a deprotonated carboxyl group. In contrast, [1,4-13C]CLA modification of Escherichia coli PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C1 chemical shifts indicate substantially deprotonated carboxylic acid groups. For one of four observed forms of [1,4-13C]CLA-modified E. coli PBGS, an analog of the P-site Schiff base is found. Bovine and E. coli PBGS contain two different zincs, ZnA and ZnB. Past results placed ZnA near A-side ALA. [1,4-13C]CLA modifies E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine cluster implicated in binding ZnB. This result places ZnB near P-side ALA. E. coli PBGS binds a third type of divalent metal, MgC or MnC, which is found to have no significant effect on the 13C NMR spectrum of the [1,4-13C]CLA-modified protein.
Jaffe
E K
EK
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Volin
M
M
Myers
C B
CB
Abrams
W R
WR
D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000031:analogs & derivatives; D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology; D007982:Levulinic Acids / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D002247:Carbon Isotopes; D007982:Levulinic Acids; D008667:Metalloproteins; C013439:5-chloro-4-oxopentanoic acid; D000622:Aminolevulinic Acid; D000623:Porphobilinogen Synthase; D015032:Zinc