STEADY-STATE KINETIC-STUDIES WITH THE POLYSULFONATE U-9843, AN HIV REVERSE-TRANSCRIPTASE INHIBITOR

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor o f HIV-1 RT and possesses excellent antiviral activity at nontoxic dos es in HIV-1 infected lymphocytes grown in tissue culture. Kinetic stud ies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity wer e carried out in order to determine if the inhibitor interacts with th e template:primer or the deoxyribonucleotide triphosphate (dNTP) bindi ng sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and it s substrates, proved inadequate for the analysis of the experimental d ata. The data were thus analyzed using steady-state Briggs-Haldane kin etics assuming that the template:primer binds to the enzyme first, fol lowed by the binding of the dNTP and that the polymerase is a processi ve enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its su bstrates and the inhibitor. The calculated rate constants are in agree ment with this model and the results indicated that U-9843 acts as a n oncompetitive inhibitor with respect to both the template:primer and d NTP binding sites. Hence, U-9843 exhibits the same binding affinity fo r the free enzyme as for the enzyme-substrate complexes and must inhib it the RT polymerase by interacting with a site distinct from the subs trate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.