Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
Role of methionine 184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis.
Biochemistry. 1996 Feb 20;35(7):2168-79. Unique Identifier : AIDSLINE MED/96194137 Pandey VN; Kaushik N; Rege N; Sarafianos SG; Yadav PN; Modak MJ; Department of Biochemistry and Molecular Biology, UMD-New Jersey; Medical School, Newark 07103, USA.
Abstract:
Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.
Keywords: Amino Acid Sequence Base Sequence DNA Polymerases/*METABOLISM DNA Primers DNA Replication/*GENETICS HIV-1/*ENZYMOLOGY Kinetics Methionine/*METABOLISM Molecular Sequence Data Mutagenesis, Site-Directed Nucleic Acid Heteroduplexes RNA-Directed DNA Polymerase/GENETICS/ISOLATION & PURIF/ *METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Templates JOURNAL ARTICLE 961030
M96A1457
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Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
