Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein.
Upon renaturation, the polyprotein MBP-delta TF-Protease-delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (delta TF) and the polymerase (delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps. The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-delta Pol, which has enzymatic activity comparable to that of the mature enzyme. Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus. The rate of increase in enzymatic activity is identical to that of the appearance of MBP-delta TF and the disappearance of the MBP-delta TF-Protease-delta Pol. Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration. 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 which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.
Louis
J M
JM
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Nashed
N T
NT
Parris
K D
KD
Kimmel
A R
AR
Jerina
D M
DM
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015685:Fusion Proteins, gag-pol / Q000378:metabolism*; D016333:HIV Protease / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D015685:Fusion Proteins, gag-pol; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides; D010436:Pepstatins; C461599:maltose transport system, E coli; C014195:Streptomyces pepsin inhibitor; D016333:HIV Protease; C031375:pepstatin