Pyridoxal-5'-phosphate inhibits the polymerase activity of a recombinant RNAase H-deficient mutant of HIV-1 reverse transcriptase.
We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.
Moen
L K
LK
Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529-0126.
Bathurst
I C
IC
Barr
P J
PJ
D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*
D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D011732:Pyridoxal Phosphate; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H; D008274:Magnesium