Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Amphipatic molecules affect the kinetic profile of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Dioxygenases are nonheme iron enzymes that biodegrade recalcitrant compounds, such as catechol and derivatives, released into the environment by modern industry. Intradiol dioxygenases have attracted much attention due to the interest in their use for bioremediation, which has demanded efforts towards understanding their action mechanism and also how to control it. The role of unexpected amphipatic molecules, observed in crystal structures of intradiol dioxygenases, during catalysis has been poorly explored. We report results obtained with the intradiol enzyme chlorocatechol 1,2-dioxygenase (1,2- CCD) from Pseudomonas putida subjected to delipidation. The delipidated enzyme is more stable and shows more cooperative thermal denaturation. The kinetics changes from Michaelis-Menten to a cooperative scheme, indicating that conformational changes propagate between monomers in the absence of amphipatic molecules. Furthermore, these molecules inhibit catalysis, yielding lower vmax values. To the best of our knowledge, this is the first report concerning the effects of amphipatic molecules on 1,2-CCD function.FAPESPCNPqCAPE

Low-temperature EPR spectra of ferricyt <i>c</i> treated with AA.

<p>(A) EPR spectra of (300 µM) ferricyt <i>c</i> after a 12-h incubation with (1.0–5.0 mM) AA. (B) Time-response ERP spectra of ferricyt <i>c</i> treated with (1.0 mM) AA for 30 min. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C. (C) MCD spectra of ferricyt <i>c</i> in the presence of AA. The conditions are ferricyt <i>c</i> (40 µM) MCD spectrum before treatment with AA (thick line) and MCD ferricyt <i>c</i> spectrum after 12 h of (5.0 mM) AA treatment (thin line). (D) CD spectrum of ferricyt <i>c</i> treated with 5.0 mM AA. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C.</p

Proposed mechanism of AA oxidation catalyzed by iron and copper ions (Adapted from Dutra et al.[14]).

<p>Proposed mechanism of AA oxidation catalyzed by iron and copper ions (Adapted from Dutra et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057790#pone.0057790-Dutra1" target="_blank">[14]</a>).</p

EPR spin-trapping studies of the ferricyt <i>c</i>/AA system under aerobic conditions.

<p>EPR spectra of DMPO-radical adducts were obtained after a 4-min incubation of 15 mM AA at 25°C in 50 mM phosphate buffer (pH 7.4) with (25 mM) DMPO: (A) DMPO experiments, (B) DMPO in the presence of DMSO 30% v/v, (C) DMPO in the presence of ethanol 30% v/v. For all of the figures: (a) control with ferricyt <i>c</i> (150 µM); (b) AA (15mM); (c) AA (15 mM)+desferoxamine (100 µM); (d) ferricyt <i>c</i> (150 µM)+AA (15 mM); (e) system d+CuZnSOD (50 U/mL); (f) system d+catalase (15 µM) for Fig. 2A and 2C only. Instrumental conditions: microwave power, 20.2 mW; modulation amplitude, 1.0; time constant, 1.63 s; scan rate 0.1 G/s; and receiver gain, 1.12×106.</p

Oxygen uptake by AA in the presence of ferricyt <i>c</i>.

<p>Experimental conditions: (50 µM) ferricyt <i>c</i> in the presence or absence of (5.0 mM) AA in 50 mM phosphate buffer, pH 7.4, at 37°C for 30 min. Experiments were performed in the absence or presence of catalase (5.0 µM) or CuZnSOD (50 U/mL). Data are representative of five independent runs. *p<0.05 relative to the system containing only AA and #p<0.05 relative to the AA/ferricyt <i>c</i> system.</p

EPR spin-trapping studies and computer simulation of the AA system in the presence and absence of ferricyt <i>c</i>, under aerobic conditions.

<p>EPR spectra of DMPO-radical adducts were obtained after a 4-min incubation of (15 mM) AA at 25°C with (150 µM) cyt <i>c</i> in 50 mM phosphate buffer (pH 7.4) with (400 mM) DMPO. (A) Experimental spectrum (trace a) and computer simulations (traces b-d) of the DMPO/AA system, (B) Experimental spectrum (trace a) and computer simulations of the DMPO/AA/cyt <i>c</i> system (traces b-e). Trace <i>c</i> in panels A and B represents the DMPO-<b>^•</b>OH adduct spectrum, and trace d can attributable to the DMPO-AA<b>^•</b> adduct. Trace e in panel B represents an unknown DMPO adduct. Instrumental conditions: microwave power, 20.2 mW; modulation amplitude, 1.0; time constant, 1.63 s; scan rate 0.1 G/s; and receiver gain, 1.12×106.</p

The effect of heparin on steady-state FRET-peptide hydrolysis by HNE.

<p>The influence of heparin concentration upon steady-state HNE kinetics parameters <i>k</i>_cat [A] and <i>K</i>_M [B] was determined spectrofluometricallyas described under “Experimental Procedures”. The fluorescence increase of FRET-peptide hydrolysis (0.2 - 10 µM) by 12.6 nM HNE were performed in 10 mM Hepes, pH 7.4, containing 140 mM NaCl and 0,05% Triton X-100 at 37°C.</p

Representation of the complex of both FRET-peptide and heparin with HNE.

<p><i>A</i>, Docking of HNE with the substrate AMESVMGYFHRSQ,the secondary structure elements of the HNE are represented by <i>indigo blue schematics</i> (<i>arrows</i> for extended strands, and <i>cylinders</i> for helices).The carbon atoms of the substrate are indicated by a <i>green sticks</i>, view of the minimum energy conformation from docking, showing the substrate labeled at P1Val and P1'Met completely engulfed inside the HNE active site cavity: S195, H57 and D103 (dark blue for nitrogen atoms, light yellow for carbon atoms, red for oxygen atoms, grey for hydrogen atoms and dark yellow for sulfur). HNE active site residues are labeled following the numbering of chymotrypsin. <i>B</i>, Docking of HNE with heparin,the secondary structure elements of the HNE are represented by <i>green schematics</i> (<i>arrows</i> for extended strands, and <i>cylinders</i> for helices) and the catalytic residues of the HNE are labeled.The carbon atoms of heparin chain are indicated by a <i>cyan sticks</i> and its sulfur atoms are indicated by dark yellow. <i>C</i>, Enlarged view of the HNE catalytic residues S195, H57 and D103 with substrate labeled at P1Val and P1'Met. <i>D</i>, Stereo view of the ternary complex between HNE•Heparin•Substrate, the secondary structure elements of the HNE are represented by <i>green schematics</i>. The carbon atoms of the substrate are indicated by a <i>cyan sticks</i> and the carbon atoms of heparin are indicated by <i>magenta sticks</i>.</p

Stopped-flow time-tracesofFRET-peptidehydrolysis by HNE.

<p><i>A</i><b>,</b> Stopped-flow fluorescence kinetic recording of 3.8 µMFRET-peptide hydrolysis by 12.6 nM HNEat 25°C in 10 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl. The progress of the reaction was monitored by the fluorescence increase of the released product recorded on 2 adjacent time regions with distinct sampling periods: 0.5 ms from 0 to 2 s, 2 ms from 2 to 6 s. Gray solid line represents the best fit obtained from the mechanism depicted in Scheme I in the absence of heparin with the aid of DynaFit IV® software (see Experimental Procedures). The insert graphic represents the associate residual errors from the best fit curve with experimental data. <i>B</i>, the HNE species as a function of time reaction: free enzyme, E (<b>–</b>); complex enzyme-substrate, ES <b>(–</b> •<b>–</b>) and acyl-enzyme, ES' (<b>- - -</b>).</p