title: Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.
abstract: 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.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Fenstermacher
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: DeStefano
mesh_terms: D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D055162:Biocatalysis / Q000187:drug effects*; D002118:Calcium / Q000494:pharmacology; D017931:DNA Primers / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability / Q000187:drug effects; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009154:Mutation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic; D014779:Virus Replication / Q000187:drug effects; D015032:Zinc / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D003854:Deoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus; D008274:Magnesium; D015032:Zinc; D002118:Calcium