The purified 52 kDa polyprotein (MBP-12AA-Protease-19AA) containing 12 amino acids of the transframe protein and 19 amino acids of the reverse transcriptase flanking the N and C terminus of the HIV-1 protease, respectively, upon 10 fold dilution, undergoes time dependent autoprocessing to release the 11 kDa mature protease in two steps. The initial step involves the cleavage at the N terminus of the protease to produce the protein species MBP-12AA (39 kDa) and Protease-19AA (13.2 kDa), followed by a slower cleavage of the 13.2 kDa protease intermediate leading to the release of the 11 kDa mature protease. The inhibition of the autoprocessing reaction with a peptide substrate of the mature protease and pepstatin A, a specific inhibitor of aspartic acid proteases indicates that the active site of the dimeric protease is fully formed in the renatured polyprotein. The autoprocessing reaction also requires a catalytic group with an apparent pK(a) of 6.7 in its protonated form, as does the hydrolysis of peptide substrates by the mature protease. The fragmentation of the polyprotein is concomitant with an increase in protease activity. Initial rates of the first-step of the reaction are linearly dependent on the protein concentration indicating that the reaction is first-order in protein concentration monitored by following the disappearance of the polyprotein and appearances of the MBP-12AA and enzymatic activity. The first-order rate constant for the slowest step on the protein folding pathway is 15 times faster than the first cleavage on the autoprocessing pathway. Thus the folding and dimerization of the polyprotein is not a rate limiting step in the autoprocessing reaction. The rate of appearance of enzymatic activity is identical to that of the appearance of MBP-12AA and to the disappearance of the MBP-12AA-Protease-19AA and to the sum of the accumulation of the 13.2 kDa Protease-19AA and 11 kDa protease. This indicates that the 13.2 kDa protein has enzymatic activity comparable to that of the mature 11 kDa protease. These results are discussed in the framework of a mechanism in which homodimerization of the protease domains of the fusion protein must form prior to the rate-limiting intramolecular cleavage of the N-terminal strands leading to the appearance of mature like protease activity. We have also characterized novel protease cleavage sites within the 72 amino acid nucleocapsid precursor (p7/p1) and the first zinc binding domain of the mature 55 amino acid nucleocapsid protein (p7). The kinetic parameters of these cleavages were determined. The rate of hydrolysis of the p7/p1 scissile bond was not affected by the conformation of the p7 domain induced as a result of zinc binding, whereas, the cleavage within the p7 protein was inhibited when complexed with zinc.
