S1 nuclease: immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site.
A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.
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Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Shankar
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D000818:Animals; D000906:Antibodies; D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D002020:Buffaloes; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003545:Cysteine; D004247:DNA / Q000378:metabolism; D005779:Immunodiffusion; D007700:Kinetics; D008099:Liver; D012313:RNA / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000494:pharmacology
D000906:Antibodies; D013439:Sulfhydryl Reagents; D012313:RNA; D009764:o-Phthalaldehyde; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D003545:Cysteine