We continued detailed biochemical studies on mechanisms of action of mammalian DNA replication proteins. Steady-state kinetic analysis of the HIV reverse transcriptase confirmed an overall kinetic scheme for the reverse transcriptase mechanism proposed earlier, and properties of the first two steps in the proposed reaction pathway were studied. A DNA segment containing the coding region for this enzyme was subcloned into an expression vector, and the enzyme and 4 subfragments were overproduced from E. coli and purified. These recombinant enzymes and proteins are under study. HIV RT works in the cell as a complex of two protein molecules. Compounds that block interaction between these two proteins may prove to be highly specific inhibitors of the RT. We have identified a small region in the RT that is responsible for this interaction and have shown that a peptide inhibitor can block formation of the normal complex. Possible use of this peptide inhibitor as an antiviral agent is under study. Finally, we found that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis (K-i = 20 nM) against substrate dTTP in the poly[r(A)].oligo- [d(T)] system. Kinetic studies of DNA synthesis using AZTTP as substrate revealed that the Km is 3 (mu)M. Using these values, a method was devised for calculation of rate constants for the enzyme-poly[r(A)].oligo[d(T)] association (kon = 2.6 x 108 M-1 s-1) and dissociation (koff= 39s-1), along with a KD value for dTTP binding (about 180 nM).
