Human immunodeficiency virus type 1 reverse transcriptase. 3'-Azidodeoxythymidine 5'-triphosphate inhibition...

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) is crucial for viral replication.
• HIV-1 RT catalyzes DNA synthesis through an ordered sequential mechanism involving template-primer (T.P) and deoxynucleoside triphosphate binding.
• Catalytic cycling can be limited by conformational changes or enzyme release from the T.P complex.

Purpose of the Study:

• To investigate the rate-limiting steps in HIV-1 RT catalysis.
• To determine kinetic parameters for single-nucleotide incorporation using chain-terminating substrates.
• To explore the influence of template-primer dissociation on enzyme kinetics.

Main Methods:

• Kinetic analysis of single-nucleotide incorporation by HXB2R HIV-1 RT using 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate.
• Utilized a poly(rA)-oligo(dT)16 template-primer system.
• Examined inhibition of processive deoxythymidine monophosphate incorporation by chain-terminating substrates.

Main Results:

• The time course of AZTMP incorporation was biphasic, with kcat (0.42 min-1) similar to the rate constant for RT-T.P complex dissociation.
• Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM).
• No simple correlation was observed between the RT-T.P dissociation rate constant and AZTTP inhibition constant (Ki,AZTTP).

Conclusions:

• A simple ordered model for single-nucleotide incorporation by HIV-1 RT is inadequate.
• Different forms of the RT-T.P complex likely exist and can limit catalytic rates.
• A two-step binding mechanism for T.P, involving isomerization before deoxynucleotide binding, may better explain the observed kinetics.