Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.
A single-chain tethered dimer of human immunodeficiency virus protease (HIV-PR) was produced by expression of a synthetic gene in Escherichia coli. The tethered dimer, which consists of two 99-amino acid HIV-PR subunits linked together by a pentapeptide, was isolated from inclusion bodies and refolded as an active protease with enzymatic properties very similar to those of the natural dimer at pH 5.5. In addition to demonstrating that the tethered dimer is active, we have shown that the tethered dimer is more stable than the natural HIV-PR dimer at pH 7.0. This is attributed to dissociation of the natural HIV-PR dimer, for which a surprisingly high dissociation constant, 5 X 10(-8) M was measured. Furthermore, the tethered dimer offers an opportunity to produce asymmetric dimer mutants and thereby determine the effect of changes in one of the two subunits on protease activity. In one such mutant, a single active-site aspartic residue was changed to a glycine residue. This protein was inactive, consistent with a requirement for an aspartic residue from each subunit to constitute an active site of HIV-PR.
Cheng
Y S
YS
Central Research and Development Department, E.I. du Pont de Nemours and Co., Wilmington, DE 19880-0328.
Yin
F H
FH
Foundling
S
S
Blomstrom
D
D
Kettner
C A
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006678:HIV / Q000201:enzymology / Q000235:genetics*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000138:chemical synthesis; D013379:Substrate Specificity
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D016333:HIV Protease