Human immunodeficiency virus type-1 reverse transcriptase. Contribution of Met-184 to binding of nucleoside 5'-triphosphate

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those...

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Vydané v:The Journal of biological chemistry Ročník 271; číslo 23; s. 13656
Hlavní autori: Wilson, J E, Aulabaugh, A, Caligan, B, McPherson, S, Wakefield, J K, Jablonski, S, Morrow, C D, Reardon, J E, Furman, P A
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States 07.06.1996
Predmet:
Amino Acid Sequence
Base Sequence
Binding Sites - genetics
Conserved Sequence
DNA Primers - genetics
HIV Reverse Transcriptase
HIV-1 - enzymology
HIV-1 - genetics
Humans
In Vitro Techniques
Kinetics
Molecular Sequence Data
Nucleotides - metabolism
Point Mutation
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
RNA-Directed DNA Polymerase - genetics
RNA-Directed DNA Polymerase - metabolism
Substrate Specificity
ISSN:0021-9258
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Shrnutí:Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the kpvalues of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5'-triphosphate.
Bibliografia:ObjectType-Article-1
SourceType-Scholarly Journals-1
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content type line 23
ISSN:0021-9258
DOI:10.1074/jbc.271.23.13656