title: Conformational changes of HIV reverse transcriptase subunits on formation of the heterodimer: correlation with kcat and Km.
abstract: The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) is initially expressed as a 66-kDa protein and is subsequently proteolytically processed in vivo to form a 66-kDa/51-kDa heterodimer. Comparison of circular dichroism spectra of the 66-kDa, 51-kDa, and heterodimeric forms of RT indicates that the conversion is accompanied by dramatic changes in subunit conformation. The mean residue ellipticity per subunit at 220 nm decreases from -10.7 x 10(3) deg cm2 dmol-1 for the 66-kDa protein to -6 x 10(3) deg cm2 dmol-1 for the heterodimer. The same loss of ellipticity is observed whether the heterodimer is produced by proteolysis or by mixing a separately-expressed cloned 51-kDa subunit with the 66-kDa protein. Comparison with the spectrum of the cloned 51-kDa protein suggests that much of the conformational change arises from formation of the 51-kDa subunit but substantial changes occur in the remaining 66-kDa subunit as well. A kinetic analysis was performed to correlate these conformational changes with changes in enzyme function. Application of an integrated Michaelis-Menten equation to the catalysis of poly(dT) formation using a d(pT)20-poly(rA) primer-template shows that the kcat for the heterodimer is approximately half that of the 66 kDa enzyme, decreasing from 2.9 to 1.2 nucleotides/s upon formation of the heterodimer. However, km values for the primer-template decrease from 0.54 to 0.12 microM upon heterodimer formation. Thus, kcat/Km is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
authors.forename: S F
authors.initials: SF
authors.lastname: Anderson
authors.forename: J E
authors.initials: JE
authors.lastname: Coleman
mesh_terms: D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009994:Osmolar Concentration; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D014357:Trypsin