Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90

Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):577-83. doi: 10.1042/bj2960577.


Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Benzophenones
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents
  • Cysteine / analysis*
  • Cysteine / physiology
  • DNA Primers / metabolism*
  • Enzyme Stability
  • Hot Temperature
  • Leukemia Virus, Murine / enzymology*
  • Maleimides
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Mapping
  • RNA-Directed DNA Polymerase / chemistry*
  • RNA-Directed DNA Polymerase / metabolism
  • Reverse Transcriptase Inhibitors
  • Sulfhydryl Compounds / analysis*
  • Templates, Genetic
  • Trypsin
  • Ultraviolet Rays


  • Benzophenones
  • Cross-Linking Reagents
  • DNA Primers
  • Maleimides
  • Reverse Transcriptase Inhibitors
  • Sulfhydryl Compounds
  • benzophenone-4-maleimide
  • RNA-Directed DNA Polymerase
  • Trypsin
  • Cysteine