Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification.

Biochemistry (Impact Factor: 3.02). 01/2013; 52(5). DOI: 10.1021/bi301643h
Source: PubMed


Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl274)-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5'-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Lys167 to cysteine crippled the DNA cleavage and religation transesterification steps (kcl = 4.3 x 10-4 s-1; krel = 9 x 10-4 s-1). The transesterification activities of the K167C enzyme were revived by in vitro alkylation with 2-bromoethylamine (kcl = 0.031 s-1; krel ≥ 0.4 s-1) and 3-bromopropylamine (kcl = 0.013 s-1; krel = 0.22 s-1), which convert the cysteine to γ-thialysine and γ-thiahomolysine, respectively. These chemically installed lysine analogs were more effective than a genetically programmed arginine-167 substitution characterized previously. The modest differences in the transesterification rates of the 2-bromoethylamine and 3-bromopropylamine-treated enzymes highlights that TopIB is tolerant of a longer homolysine side chain for assembly of the active site and formation of the transition state.

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    ABSTRACT: The protonation states of the two active-site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S-aminoethylated cysteine (γ-thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd-carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free-base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pKa values were compared to the pKa values calculated computationally, using molecular-dynamics simulations and a thermodynamic cycle. Study of the γ-thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half-reactions. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
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