Kinetic properties of HIV-1 protease produced by total chemical synthesis with cysteine residues replaced by isosteric L-?-amino-n-butyric acid

Abstract

Human immunodeficiency virus-I protease, produced by total chemical synthesis with the cysteine residues replaced by L-a-amino-n-butyric acid ([Aba 67' 95] HIV-1 PR), has been used extensively for the X-ray crystallographic structural analysis of the enzyme and its complexes utilized in drug design. Here we report kinetic studies on the synthetic enzyme. The pH optimum is 5.5 at ionic strengths of 0.1 and 1.0. The acid pH optimum is due to a decrease in binding affinity at higher pH values rather than to a reduction in catalytic efficiency. Activity is markedly increased by high ionic strength, although the major effect is on Km and not kca t. The effect of pH and ionic strength on the kinetic constants determined for substrates and inhibitors is demonstrated and attention is drawn to the need for assay conditions to be explicitly reported in studies on inhibitor activity. The effect of a number of inhibitors has been measured against the synthetic enzyme and a recombinant HIV-1 PR. This work shows that [Aba 67' 95] HIV-1 PR has full enzymatic activity and normal kinetic properties.

Molecular Recognition of Macrocyclic Peptidomimetic Inhibitors by HIV-1 Protease,

“…The two cysteine residues (Cys67 and Cys95) of the native sequence were replaced with the isosteric analogue L-Ramino-n-butyric acid (Aba) for ease of synthesis and handling to yield [Aba 67,95 ]HIVPR (7). This enzyme was fully active and had the same kinetic properties as the cloned enzyme (19). [Aba 67,95 ]HIVPR, which we refer to as HIVPR, was used in the HIVPR-1 cocrystallization experiment.…”

“…Synthesis of the macrocyclic peptidomimetic inhibitors was carried out as described previously ( − ). [Aba 67,95 ]HIVPR (SF2 isolate) and [Aba 67,95 ]HIVKI were chemically synthesized in our laboratories by solid phase peptide synthesis ( , ). The two cysteine residues (Cys67 and Cys95) of the native sequence were replaced with the isosteric analogue l -α-amino- n -butyric acid (Aba) for ease of synthesis and handling to yield [Aba 67,95 ]HIVPR ().…”

“…HIVPR Inhibition Assay. Enzyme activity was measured using a continuous fluorimetric assay described previously ( , ). Inhibition of HIVPR was assessed using the following conditions: 37 °C, 100 mM MES buffer at pH 6.5 [containing 10% (v/v) glycerol and 50 μg/mL bovine serum albumin], and 50 μM substrate [2-(aminobenzoyl)-Thr-Ile-Nle-Phe( p -NO 2 )-Gln-Arg-NH 2 ].…”

A Continuous Fluorometric Assay for the Feline Immunodeficiency Virus Protease

“…Side chain protection of appropriate lysine DMSO in the assay was only 1% (v/v). It has been reported that DMSO concentrations õ2% have little efresidues with a base labile fluorenylmethoxycarbonly group during peptide synthesis permitted their selec-fect on the catalytic efficiency of the HIV PR (18). It has been reported that Abz derivatives in which the tive deprotection (2 1 20% v/v piperidine in DMF for 5 min) for reaction with HBTU-activated 2-aminobenzoic Abz group is attached to the epsilon amine of a lysine…”

Substrate-Based Cyclic Peptidomimetics of Phe-Ile-Val That Inhibit HIV-1 Protease Using a Novel Enzyme-Binding Mode

“…Table reports inhibitor potencies of some molecules containing the macrocyclic mimetic of Phe-Ile-Val against synthetic HIVPR in vitro. Under the assay conditions, reference inhibitors JG365 3 and DM323 potently inhibit cleavage of a synthetic fluorogenic substrate . The larger 16-membered ring in 5 was no more effective than the 15-membered ring of 4 , and neither ring on its own was a significant inhibitor (IC 50 ∼ 1−10 μM).…”