Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Berit Olsen Krogh, Stewart Shuman
2001 Journal of Biological Chemistry  
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 (Tyr-274) 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
more » ... 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. Type IB DNA topoisomerases relax DNA supercoils via a reaction pathway entailing noncovalent binding of the enzyme to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3Ј-phosphotyrosyl)-protein intermediate, strand passage, and strand religation (1, 2). Tyrosine recombinases use a similar transesterification mechanism to form and resolve Holliday junctions. The catalytic domains of topo 1 IB and tyrosine recombinases adopt a common fold composed of eight ␣ helices and a three-stranded antiparallel ␤ sheet (3-9). The constituents of the active site occupy similar positions in the topo IB and recombinase tertiary structures. Four conserved amino acid side chains (e.g. Arg-130, Lys-167, Arg-223, and His-265 in the vaccinia topoisomerase) cat-alyze the attack of the active site tyrosine nucleophile (Tyr-274) on the scissile phosphodiester (10 -12). Mutational, stereochemical, and structural data for vaccinia and nuclear topoisomerase IB and tyrosine recombinases suggest that the two arginines and the histidine contact the nonbridging oxygens of the scissile phosphodiester and that these interactions serve to stabilize a proposed pentacoordinate phosphorane transition state (3, 5, 9, 10 -13). Recently we used 5Ј-bridging phosphorothiolate-modified DNAs to implicate Lys-167 of vaccinia topoisomerase as a general acid catalyst of the DNA cleavage reaction (14) . The hypothesis was that if the expulsion of the 5Ј-oxygen of the leaving DNA strand was indeed catalyzed by a general acid on the topoisomerase, then the requirement for the general acid ought to be alleviated by introducing a 5Ј-bridging phosphorothiolate at the scissile phosphodiester because the pK a of the DNA 5Ј-SH is ϳ5 log units lower than that of the DNA 5Ј-OH. Accordingly the diagnostic feature of a topoisomerase mutant defective in general acid catalysis would be a significant increase in the cleavage rate on a 5Ј-S substrate compared with a 5Ј-O substrate. The "sulfur/oxygen ratio" ought not to increase for topoisomerase mutants that are impaired in other aspects of transesterification chemistry (e.g. transition state stabilization). Our findings were that that the 5Ј-S group restored activity to the catalytically defective K167A mutant, whereas there was no positive thiolate effect for mutants R223A and H265A (14). Thus, we concluded that Lys-167 functions in proton transfer to the leaving strand. Lys-167 is located in a flexible interstrand hairpin loop of vaccinia topoisomerase and is conserved in all known type IB topoisomerases and tyrosine recombinases. Our earlier experiments did not address whether Arg-130 plays any role in leaving strand expulsion. Arg-130, which is invariant among type IB topoisomerases and tyrosine recombinases, enhances the transesterification rate of vaccinia topoisomerase by 5 orders of magnitude as gauged by the effect of the R130A mutation (10). Because a conservative R130K substitution had little restorative effect, it was surmised that multivalent contacts of the guanidinium nitrogens of Arg-130 with the scissile phosphodiester are essential for catalysis. The occurrence of either one or two hydrogen bonding contacts between this conserved arginine side chain and the DNA backbone in various crystal structures of topoisomerase IB and tyrosine recombinases (3, 5, 9) was broadly consonant with the mutational data for the vaccinia enzyme (11). Here we report the surprising observation that Arg-130 plays a major role in proton transfer to the 5Ј-leaving DNA strand. EXPERIMENTAL PROCEDURES Topoisomerase Mutants-Recombinant wild-type vaccinia topoisomerase and mutants K167A, R130A, and R130K were produced in Escherichia coli and purified by phosphocellulose chromatography as described previously (15). The double mutant R130K/K167A was constructed for the present study and was produced and purified using the same protocol. Equilibrium Cleavage Assay-Reaction mixtures containing (per 20 l) 50 mM Tris-HCl (pH 7.5), 0.3 pmol of 34-mer/60-mer DNA (Fig. 1) , and 75 ng of purified topoisomerase were incubated at 37°C. Aliquots (20 l) were withdrawn at the times indicated and quenched immediately with SDS. The mixtures were digested with 10 g of proteinase K for 60 min at 37°C, then adjusted to 50% formamide, and heat-denatured. The samples were analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in 90 mM Tris borate, 2.5 mM
doi:10.1074/jbc.c100681200 pmid:11756402 fatcat:6vuhhhyrgjhlvn2zatbac23ylu