MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

酶学，动力学和合成，物理和分析化学

Enzymology, kinetics and synthetic, physical and analytical chemistry are used in mechanistic investigations of the reverse transcriptase and protease of human immunodeficiency virus type 1 (HIV-1), with the ultimate goal of developing specific inhibitors for these enzymes. i) In a previous report, we described the kinetics of proteolytic steps in the autoprocessing of the HIV-1 protease from a construct containing the protease sequence flanked by a 19-amino acid Pol sequence at the C- terminus and a 12-amino acid sequence from the trans frame peptide at the N-terminus, which is fused to the maltose binding protein of E. coli. We have now examined, by fluorescence spectroscopy, the renaturation of the fusion protein that initiates autoprocessing. Two processes were observed: an initial step (or steps) whose rate was too fast to measure using conventional mixing techniques, and a slower step that was attributed to cis/trans isomerization of peptide bonds involving the imino nitrogen of proline. The latter step is ca. 15 times faster than the initial proteolytic cleavage at the amino terminus of the protease sequence. This proteolytic step occurs both intramolecularly with the dimeric fusion protein alone, and intermolecularly in the presence of mature HIV-1 protease. Despite the low catalytic activity of the dimeric fusion protein, a fairly high concentration (1.4 fM) of mature protease is required to give a rate that is competitive with intramolecular cleavage, as a result of the entropic advantage of intramolecularity. The 13.2-kDa protein intermediate that is formed by cleavage at the amino terminus of the protease sequence has been purified to >90% homogeneity. Preliminary results suggest that the dissociation constant for the dimeric form of this protein is much larger than that for either the full length fusion protein or the mature protease. ii) Kinetics of nonprocessive incor- poration of 4-thiothymidine into a DNA template-primer by the Klenow fragment of E. coli DNA polymerase I were measured spectrophotometrically at 335 nm. The rate constant for dissociation of the oligonucleotide product from the enzyme is ca. 6 times larger than the corresponding rate constant for its thymidine-containing analog.