Sensitive, soluble chromogenic substrates for HIV-1 proteinase.
By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.
Richards
A D
AD
Department of Biochemistry, University of Wales College of Cardiff, United Kingdom.
Phylip
L H
LH
Farmerie
W G
WG
Scarborough
P E
PE
Alvarez
A
A
Dunn
B M
BM
Hirel
P H
PH
Konvalinka
J
J
Strop
P
P
Pavlickova
L
L
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism; D015684:Gene Products, pol / Q000378:metabolism*; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives; D011480:Protease Inhibitors; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D015683:Gene Products, gag; D015684:Gene Products, pol; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; C040239:4-nitrophenylalanine; D010649:Phenylalanine; D010450:Endopeptidases; D016333:HIV Protease