Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.
The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS)
Markham
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Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Myers
C B
CB
Harris
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Volin
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M
Jaffe
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D000623:Porphobilinogen Synthase / Q000737:chemistry*; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000737:chemistry
D013113:Spin Labels; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D000623:Porphobilinogen Synthase; D003545:Cysteine