Lysine 152 of MuLV reverse transcriptase is required for the integrity of the active site?

Qingli Shi, Kamalendra Singh, Aashish Srivastava, Neerja Kaushik, Mukund J. Modak

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13 Scopus citations

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 Km·dNTP (except for that of K152E) and an ∼3-10-fold decrease in kcat 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-I 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.

Original languageEnglish (US)
Pages (from-to)14831-14842
Number of pages12
JournalBiochemistry
Volume41
Issue number50
DOIs
StatePublished - Dec 17 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry

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