EXPLOITING NUCLEOTIDE THIOPHOSPHATES TO PROBE MECHANISTIC ASPECTS OF ESCHERICHIA-COLI DNA GYRASE

The interaction of DNA gyrase with ATP has been probed using a range o f thiophosphate ATP analogs. ATP gamma S is not detectably hydrolyzed by gyrase but can support limited, probably catalytic, DNA supercoilin g. ATP gamma S is a good inhibitor of both ATP hydrolysis and ATP-supp orted supercoiling. In contrast, both ATP alpha S(R-p) and ATP beta S( R-p) have been shown to be good substrates for the ATPase reaction of gyrase and to support catalytic DNA supercoiling. The corresponding Sp diastereoisomers do not support significant levels of supercoiling an d are not readily hydrolyzed, but are shown to be reasonable inhibitor s of gyrase, For ATP alpha S(R-p), the supercoiling and ATPase activit ies appear to be tightly coupled with the thionucleotide being apparen tly a better substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis. In the case of ATP beta S(R-p), DNA supercoilin g and nucleotide hydrolysis appear to be uncoupled in that ATP beta S( R-p) is almost as good a substrate as ATP for the ATPase reaction of b oth intact gyrase and the 43 kDa GyrB fragment, whereas it only suppor ts slow DNA supercoiling; the mechanistic consequences of these observ ations are discussed in terms of a new model for energy coupling in gy rase. DNA gyrase has been shown to be capable of catalyzing DNA superc oiling in the presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2 + Or Cd2+. The pronounced diastereoselectivity seen in both the DNA su percoiling and ATPase activity with ATP alpha S and ATP beta S togethe r with evidence from the X-ray structure of the 43 kDa GyrB-ADPNP-Mg c omplex is consistent with metal ion coordination at both of these site s, and probably to the gamma-phosphoryl center during turnover. Thus, the absolute configuration of the catalytically active Mg2+-ATP comple x is likely to involve coordination to the pro-S oxygens at both P alp ha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP compl ex with the Lambda-exo configuration.