CONFORMATIONAL-CHANGES OF HIV REVERSE-TRANSCRIPTASE SUBUNITS ON FORMATION OF THE HETERODIMER - CORRELATION WITH K(CAT) AND K(M)

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 subunits 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 k(cat) 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, K(m) values for the primer-template decrease from 0.54 to 0.12-mu-M upon heterodimer formation. Thus, k(cat)/K(m) is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template. These kinetic data are compatible with a model in which half the polymerase active centers are inactivated on formation of the heterodimer, but this loss of catalytic potential is more than offset by an increase in affinity of the heterodimer for the primer-template.