Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site.
Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase is rapidly, site-specifically modified by fluorescein maleimide (FM) at this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue. In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity. It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine. The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM. The pKa's of the fluoresceins from both FM- and IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein. These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif. The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif.
Griep
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MA
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Mesman
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TN
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000494:pharmacology; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000201:enzymology*; D005452:Fluoresceins / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry
D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009711:Nucleotides; C050046:6-iodoacetamidofluorescein; C037041:fluorescein 5-maleimide; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D008274:Magnesium; D015032:Zinc; D003545:Cysteine