       Document 0100
 DOCN  M9580100
 TI    Human immunodeficiency virus type 1 reverse transcriptase.
       3'-Azidodeoxythymidine 5'-triphosphate inhibition indicates two-step
       binding for template-primer.
 DT    9506
 AU    Jaju M; Beard WA; Wilson SH; Sealy Center for Molecular Science,
       University of Texas Medical; Branch, Galveston 77555-1068, USA.
 SO    J Biol Chem. 1995 Apr 28;270(17):9740-7. Unique Identifier : AIDSLINE
       MED/95247760
 AB    Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT)
       catalyzes DNA synthesis by an ordered sequential mechanism. After
       template-primer (T.P) binds to free enzyme, the deoxynucleoside
       triphosphate to be incorporated binds to the RT and T.P binary complex
       (RTT.P). After incorporation of the bound nucleotide, catalytic cycling
       is limited either by a conformational change (for processive synthesis)
       or release of the enzyme from the extended T.P (for single-nucleotide
       incorporation). To explore cycling through these alternate rate-limiting
       steps, we determined kinetic parameters for single-nucleotide
       incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide
       substrates 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and
       dideoxythymidine triphosphate on a homopolymeric T.P system,
       poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine
       monophosphate incorporation by these chain-terminating substrates was
       also examined. Because AZTTP is a substrate, its Km should be equivalent
       to Ki, and since Km for AZTTP should be influenced by the dissociation
       rate constant for RTT.P, we examined the effect of altering RTT.P
       dissociation on AZTTP kinetic parameters. The dissociation rate constant
       was modulated by making use of different T.P substrates, viral sources
       of RT, and a mutant RT altered at a residue that perturbs T.P binding.
       As expected from earlier work, the time course of AZTMP incorporation on
       poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower
       steady-state phase representing kcat (0.42 min-1) which was similar to
       the rate constant for RTT.P dissociation. Additionally, Km for AZTTP
       (110 nM) was lower than its equilibrium dissociation constant (1200 nM).
       AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and
       incorporation of a single nucleotide were similar. However, a simple
       correlation between the RTT.P dissociation rate constant and Ki,AZTTP
       was not observed. These results indicate that a simple ordered model for
       single-nucleotide incorporation is inadequate and that different forms
       of RTT.P exist which can limit catalysis. The results are discussed in
       the context of a two-step binding reaction for T.P where the binary
       RTT.P complex undergoes an isomerization before binding of the
       deoxynucleotide substrate.
 DE    Antiviral Agents/*PHARMACOLOGY  DNA Primers/METABOLISM
       HIV-1/*ENZYMOLOGY  Kinetics  Protein Binding  Reverse
       Transcriptase/*ANTAGONISTS & INHIB/METABOLISM  Support, U.S. Gov't,
       P.H.S.  Templates  Thymine Nucleotides/METABOLISM/*PHARMACOLOGY
       Zidovudine/*ANALOGS & DERIVATIVES/METABOLISM/PHARMACOLOGY  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

