Elucidation of the Role of Arg 110 of Murine Leukemia Virus Reverse Transcriptase in the Catalytic Mechanism:  Biochemical Characterization of Its Mutant Enzymes

Abstract

Based on the projected three-dimensional equivalence of conserved amino acids in the catalytic domains of DNA polymerases, we propose Arg 110 of MuLV RT to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain evidence to support this proposition and to assess the functional importance of Arg 110, we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala, and Glu. The mutant enzymes were characterized with respect to their kinetic parameters, ability to bind template-primers, and the mode of DNA synthesis. All the three substitutions at 110 position resulted in severe loss of polymerase activity without any significant effect on the RNase H function. In spite of an approximately 1000-fold reduction in kcat of polymerase activity with three mutant enzymes, no significant reduction in the affinities for either template-primer or dNTP substrates was apparent. Mutant enzymes also did not exhibit significant sulfur elemental effect, implying that the chemical step, i.e., phosphodiester bond formation, was not defective. Examination of the mode of DNA synthesis by the mutant enzymes indicated a shift from processive to the distributive mode of synthesis. The mutants of R110 also displayed significant loss of pyrophosphorolysis activity. Furthermore, the time course of primer extension with mutant enzymes indicated severe reduction in the rates of addition of the first nucleotide and even further reduction in the addition of the second nucleotide. These results suggest that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation. Based on these results, we propose that Arg 110 of MuLV RT participates in the conformational change steps prior to and after the chemical step of polymerase reaction.

Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

“…The studied enzymes included K103A, R110A, D114N, R116K, D114N/R116K, R116L, and N119A, all of which failed to support replication or led to a delayed phenotype when tested in viruses in the experiments described here. As previously reported, K103A and R110A enzymes retain less than 10% of wild-type activity (2,3), while the remaining enzymes retain 40 to 70% of wild-type activity in the polyriboadenylate-oligodeoxyribosylthymine assay. However, despite their relatively high levels of activity on homopolymeric templates, all of the enzymes with substitutions for D114, R116, or N119 synthesized significantly less full-length product than the wild-type enzyme when assayed on an mRNA template, suggesting that they have processivity defects (25).…”

“…K103, R110, D114, R116, and N119 are highly conserved. Predictions suggest that K103 and R110 form hydrogen bonds with the incoming nucleotide (2,3,18,25). D114, R116, and N119 are involved in critical interactions of the primer-template with the fingers domain in crystal structures of the N-terminal fragment of MLV RT complexed with DNA.…”

Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

“…These include residues of the Tyr-X-Asp-Asp (YXDD) motif, the deoxyribonucleoside triphosphate (dNTP) binding site, the ␣-helix H of the thumb domain, the conserved Leu-Pro-Gln-Gly (LPQG) motif, the finger domain, and specific amino acids such as E89 and F160 in human immunodeficiency virus type 1 (HIV-1) RT (1, 3, 4, 10, 13, 16-18, 29, 35, 42, 50). The mechanism by which fidelity is maintained may encompass features in RT that involve the local geometry of the active site, the proper positioning of the template-primer complex, or the global effects attributed to different conformational states of the protein (8,18).It was previously shown that residues 110 to 116 in HIV-1 RT are located in the active site of RT and have a moderate to high solvent accessibility, suggesting that they may play a role in dNTP binding (22,44). Furthermore, extensive mutational analysis of residues Y115 and Q151 in HIV-1 RT revealed that substitution mutations at these sites could have drastic effects on substrate dNTP binding (35, 42) as well as resistance to nucleoside analogs (21,46).…”

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

“…The catalytic importance of arginine has been shown in nucleotide substrate binding (29), and it may form a catalytic triad with serine and asparagine (30). Arginine can also have structural importance, as demonstrated by its role in conformational changes in murine leukemia virus reverse transcriptase (31). Evidence supporting a putative role for arginines and histidines in the ATP sulfurylase/APS kinase reaction mechanism comes from studies of both yeast and fungal ATP sulfurylases, which are inactivated by phenylglyoxal, which modifies arginines, and by diethylpyrocarbonate, which targets histidines (32).…”