Search

The resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) involves phosphorolytic excision of chain-terminating AZT-5'-monophosphate (AZTMP). Both pyrophosphate (PPi) and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. PPi-mediated excision produces AZT-5'-triphosphate (AZTTP), which could be immediately re-used as a substrate for viral DNA chain termination. In contrast, ATP-mediated excision produces the novel compound AZT-(5')-tetraphospho-(5')-adenosine (AZTp4A). Since little is known of the interaction of AZTp4A with HIV-1 RT, we carried out kinetic and molecular modeling studies to probe this. AZTp4A was found to be a potent inhibitor of HIV-1 RT-catalyzed DNA synthesis and of both ATP- and PPi-mediated AZTMP excision. AZTp4A is in fact an excellent chain-terminating substrate for AZT-resistant RT-catalyzed DNA synthesis, better than AZTTP (k(pol)/Kd = 6.2 and 11.9 for AZTTP and AZTp4A, respectively). The affinity of AZT-resistant HIV-1 RT for AZTp4A is at least 30,000-fold greater than that for the excision substrate ATP and approximately 10-fold greater than that for AZTTP. Dissociation of newly formed AZTp4A from RT may therefore provide a significant rate-limiting step for continued HIV-1 DNA synthesis. Our studies show that the products of PPi- and ATP-mediated excision of chain-terminating AZTMP (AZTTP and AZTp4A, respectively) are both potent chain-terminating substrates for HIV-1 RT, suggesting that there is no obvious benefit to HIV using ATP instead of PPi as the excision substrate.

The electronic and steric constraints of the dopamine beta-monooxygenase (DbetaM; E.C. 1.14.17.1) active site were studied using a series of chiral bisubstrate inhibitors. The (R) and (S) enantiomers of 5-phenyl-2-thiooxazolidone were apparent bisubstrate inhibitors for DbetaM with respect to tyramine and dioxygen, but with small enantiomeric selectivity. In contrast to the substrate specificity of the enzyme, N-methylation of both inhibitors increased the potency without altering the enantiomeric selectivity. The (S) C-4-methyl substitution was more detrimental toward the inhibition potency compared to (R) C-4-methyl substitution for both the (R) and (S) series, which was also opposite of the substrate specificity of the enzyme. The high inhibition potency and apparent bisubstrate behavior of 3-phenyl-1,5-bisthioglutarimide (XVI), a probe designed to mimic two distinct binding modes for the (R) and (S) inhibitors, suggested that they may interact with the enzyme by two different modes involving both coppers in the active site. Direct support for the interaction of the thione group(s) of XVI with the reduced DbetaM copper(s) is provided by the UV-vis spectroscopic studies. The complete disappearance of the characteristic UV absorption of XVI at 336 nm in the presence of stoichiometric amounts of reduced DbetaM demonstrate that it could be an active site titrant for reduced DbetaM. The ability of the enzyme to interact with these inhibitors by more than one mode suggests that the DbetaM active site possesses high steric and electronic tolerance.