Related Experiment Videos
Lysine 152 of MuLV reverse transcriptase is required for the integrity of the active site
Qingli Shi1, Kamalendra Singh, Aashish Srivastava
1Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey Medical School, Newark 07103, USA.
Abstract:
Comparison of the three-dimensional structure of the active sites of MuLV and HIV-1 reverse transcriptases shows the presence of a lysine residue (K152) in the substrate-binding region in MuLV RT, while its equivalent position in HIV-1 RT is occupied by a glycine (G112). To investigate the role of K152 in the mechanism of the polymerase reaction catalyzed by MuLV RT, four mutant RTs, namely, K152A, K152R, K152E, and K152G, were generated and biochemically characterized. All muteins exhibited reduced polymerase activity on both RNA and DNA template-primers with K152E being the most defective. The template-primer binding affinity and the processivity of DNA synthesis, however, remained unchanged. The steady-state kinetic characterization showed little change in K(m.dNTP) (except for that of K152E) and an approximately 3-10-fold decrease in k(cat) depending upon the template-primer and mutational substitutions. The ddNTP resistance patterns were unchanged for all muteins, suggesting no participation of K152 in ddNTP recognition. The ability of individual muteins to add dNTP on the covalently cross-linked enzyme-template-primer complex was significantly decreased. These results together with the analysis of the ion pairs in the catalytic apparatus of MuLV RT suggest that K152 participates in maintaining the integrity of the active site of MuLV RT. Examination of the prepolymerase ternary complex formation showed that neither the wild type nor any of the K152 muteins of MuLV RT are capable of forming stable ternary complexes. This property is in contrast to that of HIV-1 RT, which readily forms stable ternary complexes under similar conditions. These results further indicate that the catalytic mechanism of MuLV RT is significantly different from that of HIV-1 RT, despite the presence of a number of conserved motifs and amino acid residues.
Insights
This study investigated the role of lysine 152 (K152) in Moloney murine leukemia virus reverse transcriptase (MuLV RT). Mutants showed reduced polymerase activity, suggesting K152 maintains active site integrity and impacts MuLV RT
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Murine leukemia virus (MuLV) and Human Immunodeficiency Virus type 1 (HIV-1) reverse transcriptases (RTs) are crucial enzymes in retroviral replication.
- Structural comparison reveals a lysine residue (K152) in MuLV RT's active site, absent in HIV-1 RT (replaced by glycine G112).
- The functional significance of this K152 residue in MuLV RT's polymerase activity remains largely uncharacterized.
Purpose of the Study:
- To elucidate the role of the lysine residue at position 152 (K152) in the catalytic mechanism of MuLV RT.
- To investigate the impact of K152 substitutions on MuLV RT's polymerase activity, substrate binding, and processivity.
- To compare the catalytic mechanisms of MuLV RT and HIV-1 RT concerning ternary complex formation.
Main Methods:
- Site-directed mutagenesis was used to generate four MuLV RT mutants: K152A, K152R, K152E, and K152G.
- Biochemical characterization included assays for polymerase activity, template-primer binding affinity, processivity, and steady-state kinetics (K(m.dNTP), k(cat)).
- Analysis of ddNTP resistance and covalently cross-linked enzyme-template-primer complex formation was performed.
Main Results:
- All K152 mutant MuLV RTs exhibited significantly reduced polymerase activity on both RNA and DNA template-primers, with K152E being the most defective.
- Template-primer binding affinity and DNA synthesis processivity were unaffected by K152 mutations.
- Kinetic analysis revealed decreased catalytic efficiency (k(cat)) but largely unchanged substrate affinity (K(m.dNTP)), with no alteration in ddNTP resistance patterns.
- Mutants showed a diminished ability to extend DNA synthesis on cross-linked complexes, indicating K152's role in active site integrity.
- Unlike HIV-1 RT, wild-type and mutant MuLV RTs failed to form stable prepolymerase ternary complexes.
Conclusions:
- The K152 residue in MuLV RT is crucial for maintaining active site integrity and optimal polymerase activity, but not for substrate binding or ddNTP recognition.
- The distinct inability of MuLV RT to form stable ternary complexes, contrasting with HIV-1 RT, highlights significant differences in their catalytic mechanisms.
- These findings underscore the diverse strategies employed by retroviral reverse transcriptases despite conserved structural motifs.