Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.
Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007202:Indicators and Reagents; D007555:Isoxazoles; D008670:Metals / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology
D004277:DNA, Single-Stranded; D007202:Indicators and Reagents; D007555:Isoxazoles; D008670:Metals; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D003035:Cobalt; D000249:Adenosine Monophosphate; D012313:RNA; D004492:Edetic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases; D015032:Zinc