Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase

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Abstract
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-Δ3-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 (Δm = 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 shows that at least two of the unpaired electrons are within at most 7.6 Å 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. The results indicate that binding of this substrate does not affect the environment of the majority of the nitroxides. This is consistent with our previous proposal that it is the substrate that binds only to the Zn(II) enzyme that provides ligands to the catalytic Zn(II) (Jaffe, E.K., Abrams, W.R., Kaempfen, K.X., & Harris, K.A., 1992, Biochemistry 31, 2113–2123). SL-MMTS readily forms disulfide-linked dimers. The rate of dimer formation is proportional to the hydroxide ion concentration and is independent of both buffer and SL-MMTS concentrations. This suggests a rate-limiting hydrolysis of the S-S bond of SL-MMTS followed by rapid reaction of the liberated thiol with a second molecule of SL-MMTS. Therefore, substantial care is required in the use of SL-MMTS as a protein modification reagent at even slightly alkaline pH values.