Stabilization from autoproteolysis and kinetic characterization of the human T-cell leukemia virus type 1 proteinase.
We have developed a system for expression and purification of wild-type human T-cell leukemia virus type 1 (HTLV-1) proteinase to attain sufficient quantities for structural, kinetic, and biophysical investigations. However, similar to the human immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase also undergoes autoproteolysis rapidly upon renaturation to produce two products. The site of this autoproteolytic cleavage was mapped, and a resistant HTLV-1 proteinase construct (L40I) as well as another construct, wherein the two cysteine residues were exchanged to alanines, were expressed and purified. Oligopeptide substrates representing the naturally occurring cleavage sites in HTLV-1 were good substrates of the HTLV-1 proteinase. The kinetic parameters kcat and Km were nearly identical for all the three enzymes. Although three of four peptides representing HTLV-1 proteinase cleavage sites were fairly good substrates of HIV-1 proteinase, only two of nine peptides representing HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1 proteinase, suggesting substantial differences in the specificity of the two enzymes. The large difference in the specificity of the two enzymes was also demonstrated by inhibition studies. Of the several inhibitors of HIV-1 or other retroviral proteinases that were tested on HTLV-1 proteinase, only two inhibit the enzyme with a Ki lower than 100 nM.
Louis
J M
JM
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. jmlouis@helix.nih.gov
Oroszlan
S
S
Tözsér
J
J
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D015368:Human T-lymphotropic virus 1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease