Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aspartyl protease of human immunodeficiency virus 1 (HIV-1) has been expressed in Escherichia coli at high levels, resulting in the formation of inclusion bodies which contain denatured insoluble aggregates of the protease. After solubilization of these inclusion bodies in guanidinium chloride, the protease was purified to apparent homogeneity by a single-step reverse-phase HPLC procedure. The purified, but inactive, protein was denatured in 8 M urea and refolded to produce the active protease. Enzyme activity was demonstrated against the substrate H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH, modeled after the cleavage region between residues 128 and 135 in the HIV gag polyprotein. With this substrate, a Vmax of 1.3 +/- 0.2 mumol/(min.mg) and KM of 2.0 +/- 0.3 mM were determined at pH 5.5. Pepstatin (Iva-Val-Val-Sta-Ala-Sta-OH) and substrate analogues with the Tyr-Pro residues substituted by Sta, by Phe psi [CH2N]Pro, and by Leu psi [CH(OH)CH2]Val inhibited the protease with KI values of 360 nM, 3690 nM, 3520 nM, and less than 10 nM, respectively. All were competitive inhibitors, and the tightest binding compound provided an active site titrant for the quantitative determination of enzymatically active HIV-1 protease.
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PMID:Substrate analogue inhibition and active site titration of purified recombinant HIV-1 protease. 218 16

The structure of acetyl-pepstatin has been investigated in solution by two-dimensional NMR spectroscopy and molecular modeling. The analysis of DQFCOSY, TOCSY and NOESY spectra lead to a full assignment of the -NMR signals both in DMSO-d6 and in TFE-d3:H2O 1:1. Interproton distances, dihedral angles and exchanger regimes of NH or OH protons were derived from ROESY connectivities, coupling constants and temperature dependences of the chemical shifts, respectively. Molecular modeling using the NMR distance and dihedral angle constraints obtained in DMSO-d6 yielded a model showing a well-defined structure for the N-terminal segment Ac-1 to Sta-4, but a flexible structure for the C-terminal segment. The structure was less defined in TFE-d3:H2O 1:1 and 13C T1 measurements are indicative of higher mobility. Comparison of the NMR-determined solution structure of acetyl-pepstatin with its crystal structure when bound to HIV-1 protease shows that the conformation is more extended in the complex as a result of intermolecular interactions.
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PMID:Solution structure of the HIV protease inhibitor acetyl-pepstatin as determined by NMR and molecular modeling. 917 42

A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry and reflection absorption infrared spectroscopy, respectively. When the SAM-modified gold electrode was incubated with a solution of aspartyl protease, pepsin, a decrease in both anodic and cathodic peak currents and an increase in potential difference were observed in the cyclic voltamogram of hydroquinone as a probe, whereas a coexistence of free pepstatin fragment inhibited these phenomena, indicating the specific binding of pepsin to the fragment at the exterior of the SAM. The binding rate of the enzyme to the SAM was largely dependent on the surface density of the fragment moiety in the SAM. Furthermore, when the SAM of DTUA-Pepsta(h) on a gold colloid array deposited on an amino group-modified glass plate was immersed in a pepsin solution, absorption of the glass plate at 550 nm corresponding to a localized surface plasmon resonance of the gold colloid abruptly increased and slightly red-shifted, and a further addition of pepstatin A gradually decreased the absorbance. From the increasing and decreasing profiles of absorbance, the association constant (K(assoc)) for pepsin with the fragment on the SAM was determined. Similar phenomena were observed upon immersion of the fragment-modified SAM in a solution of HIV-1 protease, suggesting a usability of the pepstatin fragment SAM for the detection and removal of the enzyme from biological fluids.
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PMID:Self-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases. 1576 61