Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a crucial enzyme for viral replication.
• Understanding the kinetic properties of HIV-1 RT, including substrate interactions, is vital for developing antiviral therapies.

Purpose of the Study:

• To investigate substrate inhibition kinetics of HIV-1 reverse transcriptase (RT) with different substrate analogs.
• To elucidate the specific subunits and mechanisms involved in substrate inhibition.

Main Methods:

• Enzyme kinetics assays were performed using purified heterodimeric and homodimeric forms of HIV-1 RT.
• Kinetic parameters, including inhibition constants (Ki), were determined for deoxythymidine triphosphate (dTTP) and deoxyguanosine triphosphate (dGTP).
• UV crosslinking experiments were utilized to identify substrate binding sites on the enzyme subunits.

Main Results:

• Substrate inhibition was observed for both dTTP and dGTP with HIV-1 RT, with apparent Ki values around 190 microM.
• dTTP-induced substrate inhibition was specific to RNA-dependent DNA polymerase activity and did not occur with DNA.DNA template-primers.
• UV crosslinking identified the p66 subunit as the primary site for dTTP binding during substrate inhibition, ruling out an allosteric mechanism involving the p51 subunit.

Conclusions:

• HIV-1 RT exhibits substrate inhibition, particularly with dTTP, which is mediated by the p66 subunit.
• The observed substrate inhibition mechanism is distinct from allosteric regulation and may have implications for drug design targeting HIV-1 replication.