Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease.
The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
C L
CL
DiIanni
L J
LJ
Davis
M K
MK
Holloway
W K
WK
Herber
P L
PL
Darke
N E
NE
Kohl
R A
RA
Dixon
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D005813:Genes, Synthetic; D005814:Genes, Viral; D016333:HIV Protease / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D015678:Viral Structural Proteins / Q000235:genetics
D003062:Codon; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D015678:Viral Structural Proteins; D016333:HIV Protease