Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity

• Published in: The Journal of biological chemistry 2011-11, Vol.286 (47), p.40433-40442
• Main Authors: Fenstermacher, Katherine J, DeStefano, Jeffrey J
• Format: Article
• Language: English
• Subjects: Avian Myeloblastosis Virus - enzymology; Biocatalysis - drug effects; Calcium - pharmacology; Deoxyribonucleotides - metabolism; DNA Primers - metabolism; Dose-Response Relationship, Drug; Enzyme Stability - drug effects; Enzymology; HIV Reverse Transcriptase - antagonists & inhibitors; HIV Reverse Transcriptase - chemistry; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1 - drug effects; HIV-1 - enzymology; HIV-1 - genetics; HIV-1 - physiology; Kinetics; Magnesium - pharmacology; Moloney murine leukemia virus - enzymology; Mutation; Reverse Transcriptase Inhibitors - pharmacology; Ribonuclease H, Human Immunodeficiency Virus - antagonists & inhibitors; Ribonuclease H, Human Immunodeficiency Virus - chemistry; Ribonuclease H, Human Immunodeficiency Virus - metabolism; Templates, Genetic; Virus Replication - drug effects; Zinc - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.289850

Several physiologically relevant cations including Ca(2+), Mn(2+), and Zn(2+) have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg(2+) ions bound to RT. We analyzed the effects of Zn(2+) on reverse transcription and compared them to Ca(2+) and Mn(2+). Using nucleotide extension efficiency as a readout, Zn(2+) showed significant inhibition of reactions with 2 mM Mg(2+), even when present at only ∼5 μM. Mn(2+) and Ca(2+) were also inhibitory but at higher concentrations. Both Mn(2+) and Zn(2+) (but not Ca(2+)) supported RT incorporation in the absence of Mg(2+) with Mn(2+) being much more efficient. The maximum extension rates with Zn(2+), Mn(2+), and Mg(2+) were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 μM, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn(2+) and Mg(2+), and single RT molecules were able to continue extension in the presence of Zn(2+) for several hours on the same template. Consistent with this result, the half-life for RT-Zn(2+)-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg(2+), indicating ∼130-fold more stable binding with Zn(2+). Essentially, the presence of Zn(2+) promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.