Proteolytic processing of HIV-1 protease precursor, kinetics and mechanism.
Previously it was demonstrated using a model precursor that processing at the N terminus of the HIV-1 protease (PR) precedes processing at its C terminus. We now show the expression, purification, and kinetics of the autoprocessing reaction of a PR precursor linked to 53 amino acids of the native flanking transframe region (DeltaTFP-p6(pol)) of Gag-Pol and containing its two native cleavage sites. The PR contains the two cysteine residues exchanged to alanines, mutations that do not alter the kinetics or the structural stability of the mature PR. DeltaTFP-p6(pol)-PR, which encompasses the known PR inhibitor sequence Glu-Asp-Leu within DeltaTFP, undergoes cleavage at the DeltaTFP/p6(pol) and p6(pol)/PR sites in two consecutive steps to produce the mature PR. Both DeltaTFP-p6(pol)-PR and p6(pol)-PR exhibit low intrinsic enzymatic activity. The appearance of the mature PR is accompanied by a large increase in catalytic activity. It follows first-order kinetics in protein concentration with a rate constant of 0.13 +/- 0.01 min(-1) in 0.1 M acetate at pH 4.8. The pH-rate profile for the observed first-order rate constant is bell-shaped with two ionizable groups of pK(a) 4.9 and 5.1. The rate constant also exhibits approximately 7-fold higher sensitivity to urea denaturation as compared with that of the mature PR, suggesting that the cleavage at the N terminus of the PR domain from the precursor leads to the stabilization of the dimeric structure.
Louis
J M
JM
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0580, USA. jmlouis@helix.nih.gov
Wondrak
E M
EM
Kimmel
A R
AR
Wingfield
P T
PT
Nashed
N T
NT
D000595:Amino Acid Sequence; D002384:Catalysis; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D016333:HIV Protease / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
D004792:Enzyme Precursors; D016333:HIV Protease