Jm. Louis et al., KINETICS AND MECHANISM OF AUTOPROCESSING OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE FROM AN ANALOG OF THE GAG-POL POLYPROTEIN, Proceedings of the National Academy of Sciences of the United Statesof America, 91(17), 1994, pp. 7970-7974
Upon renaturation, the polyprotein MBP-Delta TF-Protease-Delta Pol, co
nsisting of HIV-1 protease and short native sequences from the trans-f
rame protein (Delta TF) and the polymerase (Delta Pol) fused to the ma
ltose-binding protein (MBP) of Escherichia coli, undergoes autoprocess
ing to produce the mature protease in two steps. The initial step corr
esponds to cleavage of the N-terminal sequence to release the protein
intermediate Protease-Delta Pol, which has enzymatic activity comparab
le to that of the mature enzyme. Subsequently, the mature enzyme is fo
rmed by a slower cleavage at the C terminus. The rate of increase in e
nzymatic activity is identical to that of the appearance of MBP-Delta
TF and the disappearance of the MBP-Delta TF-Protease-Delta Pol. Initi
al rates are linearly dependent on the protein concentration, indicati
ng that the N-terminal cleavage is first-order in protein concentratio
n. The reaction is competitively inhibited by pepstatin A and has a pH
rate profile similar to that of the mature enzyme. These results and
molecular modeling studies are discussed in terms of a mechanism in wh
ich a dimeric full-length fusion protein must form prior to rate-limit
ing intramolecular cleavage of the N-terminal sequence that leads to a
n increase in enzymatic activity.