ABSTRACT
A 120 member library of peptidocalix[4]arenes was synthesized and screened for catalysis of the hydrolysis of p-nitrophenyl acetate. His-Ser-His-calix[4]arene was found to catalyze this reaction with v(0)=3.24 x 10(-8)M/s, an increase of 1520% above background and 30% above the tripeptide (His-Ser-His) alone.
Subject(s)
Bridged-Ring Compounds/chemistry , Calixarenes/chemical synthesis , Hydrolysis , Oligopeptides/chemistry , Phenols/chemical synthesis , Bridged-Ring Compounds/pharmacology , Calixarenes/pharmacology , Catalysis , Models, Chemical , Nitrophenols/chemistry , Oligopeptides/pharmacology , Phenols/pharmacology , Time FactorsSubject(s)
Aspartic Acid Endopeptidases/metabolism , Human T-lymphotropic virus 1/enzymology , Protease Inhibitors/pharmacology , Amino Acid Sequence , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/genetics , Binding Sites , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Substrate SpecificityABSTRACT
Tetraalaninecalixarene was prepared by coupling of tetraaminocalix[4]arene with alanine. It dimerizes in methanol, providing the first example of a substituted calixarene that undergoes self-association through hydrogen bonding in polar, protic solvent. The association constant in 24:1 MeOH:H2O was determined to be 29 000 M-1. Addition of arginine or lysine results in disruption of the dimer and formation of a 1:1 complex between the amino acid and the tetraalaninecalixarene. The preparation of a peptidocalixarene that associates in polar solvent opens new doors for the use of calixarenes for molecular recognition in biologically relevant environments.
ABSTRACT
Understanding the factors that affect the activity of Human T-cell Leukemia Virus type I (HTLV-I) protease is essential for the discovery of inhibitors to be used for the treatment of HTLV-I infection, but little has been reported on the protease to date. Here we report the production of HTLV-I protease in purified yields greater than 150 mg/L, determination of its extinction coefficient, and determination of the optimum conditions for cleavage of the p19/24 substrates (DABCYL)-(GABA)-PQVL-Nph-VMH-(EDANS), (DABSYL)-(GABA)-PQVL-Nph-VMH-(EDANS), and (DABSYL)-(GABA)-PQVLPVMH-(EDANS). The highest activity was found at pH 5.2-5.3 and 37 degrees C. There was no effect on activity upon change in sodium chloride concentration from 0 to 1500 mM. The values of K(m) and k(cat) for cleavage of these substrates by the protease with and without the histidine tag were determined.
