INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS-1 REVERSE-TRANSCRIPTASE BY 3'-AZIDOTHYMIDINE TRIPHOSPHATE

In the presence of oligo(dT) . poly(rA) as primer-template, 3'-azidothymidine triphosphate (N3(3')-ddTTP) is a substrate for human immunodeficiency virus 1 reverse transcriptase with an apparent K(m) value of 3.0-mu-M. This compares with an apparent K(m) for thymidine monophosphate (dTMP) incorporation of 2.5 mu-M. The apparent V(max) value for 3'-azidothymidine monophosphate (N3(3)-ddTMP) incorporation is 50 times lower than that of dTMP incorporation. Kinetic analysis of the inhibition of reverse transcriptase by N3'(3)-ddTMP shows competitive inhibition with thymidine triphosphate (dTTP) with a K(i) of 41 nM and an uncompetitive pattern of inhibition with template-primer having a K(i) of 140 nM. This indicates incorporation of the analogue into the primer and inhibition of the enzyme by formation of a dead-end complex. The 3'-azidothymidine-terminated primer-template [N3(3'-ddT-(dT)15 . poly(rA)] is a potent competitive inhibitor versus primer-template with a K(i) of 2.4 nM and shows mixed-type inhibition against dTTP with a K(i) of 8 nM. The low inhibition constant for this chain-terminated primer suggests that such oligonucleotides can act as potent inhibitors of enzyme catalysis.