Proton relay mechanism of general acid catalysis by DNA topoisomerase IB Journal Article
| Authors: | Krogh, B. O.; Shuman, S. |
| Article Title: | Proton relay mechanism of general acid catalysis by DNA topoisomerase IB |
| Abstract: | Type IB topoisomerases cleave and rejoin DNA through a DNA-(3′-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5′-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10-4.3, was stimulated 2600-fold by a 5′-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5′-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5′-O of the leaving DNA strand. |
| Keywords: | gene mutation; amino acid substitution; protein stability; dna; conserved sequence; kinetics; recombinant proteins; substrate specificity; vaccinia virus; protons; mutagenesis, site-directed; binding sites; protein tyrosine phosphatase; catalysis; amino acids; protein folding; enzymes; proton transport; lysine; hydrogen-ion concentration; arginine; transesterification; amino acid motifs; protein modification; dna topoisomerase; dna topoisomerases, type i; enzyme active site; dna degradation; transport kinetics; vaccinia; dna strand; stabilization; equilibrium constant; thionucleotides; priority journal; article; constellation |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 277 |
| Issue: | 8 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2002-02-22 |
| Start Page: | 5711 |
| End Page: | 5714 |
| Language: | English |
| DOI: | 10.1074/jbc.C100681200 |
| PUBMED: | 11756402 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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