A TRANSIENT PRECURSOR OF THE HTV-1 PROTEASE - ISOLATION, CHARACTERIZATION, AND KINETICS OF MATURATION

Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-De lta TF-PR-Delta Pol, which contains the protease linked to short nativ e flanking sequences (Delta TF and Delta Pol) fused to the maltose bin ding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Na shed, N. T., Parris, G. D., Kimmel, A. R., and Jerina, D. M. (1994) Pr oc, Natl Acad Sci. U.S.A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP- Delta TF-PR-Delta Pol protein leads to the formation of the PR-Delta P ol intermediate, which is subsequently converted to the mature proteas e by cleavage of the C-terminal strands. We now report the purificatio n and characterization of the PR-Delta Pol intermediate and the kineti cs of its processing to the mature protease. Unlike the MBP-Delta TF-P R-Delta Pol precursor, PR-Delta Pol has proteolytic activity similar t o that of the mature enzyme at pH 5.0. The pH rate profile for k(cat)/ K-m is similar to that of the mature protease above pH 4.0. Although t he PR-Delta Pol is more sensitive than the mature protease toward dena turing reagents, both the enzymatic activity and the intrinsic fluores cence of PR-Delta Pol are linearly dependent on the protein concentrat ion, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteo lytic reaction at the N terminus of the protease, the proteolytic reac tion at the C terminus of the protease is second order in protein conc entration. These results are discussed in terms of a mechanism in whic h the C-terminally located Delta Pol peptide chains are cleaved interm olecularly to release the mature protease.