Biochemical analysis of catalytically crucial aspartate mutants of human immunodeficiency virus type 1 reverse transcriptase.
In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state ternary complex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 were completely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions, respectively. A molecular model of the ternary complex based on these results is also presented.
Kaushik
N
N
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Rege
N
N
Yadav
P N
PN
Sarafianos
S G
SG
Modak
M J
MJ
Pandey
V N
VN
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010756:Phosphoric Acids / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D010756:Phosphoric Acids; D013942:Thymine Nucleotides; D011061:Poly A; D001224:Aspartic Acid; C018368:poly(rA).oligo(dT); D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate