Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with dynafit

Zhang, Daoning; Kovach, Ildiko M.; Sheehy, John P.

doi:10.1016/j.bbapap.2008.02.004

Cited by 8 publications

(13 citation statements)

References 30 publications

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“…Moreover, k cat /K m for thrombin-catalyzed hydrolysis of S-2238 at pH 8.0 and 25 °C is 2.75 × 10 7 M −1 s −1 , (70–71) and nearly identical to k i /K i = 2.2 × 10 7 M −1 s −1 for thrombin inhibition by PPACK under identical conditions. The encounter between thrombin and S-2238 is also known, it is k 1 = 1.0 × 10 8 M −1 s −1 , which may include a conformational change.…”

Section: Results From Kinetic Studiesmentioning

confidence: 72%

“…(70–71) The tripeptide segment of the two structures are nearly identical as Pip is a Pro analog. Although K m is a complicated constant with contributions from rate constants for elementary chemical steps, its numerical value is identical to the dissociation constant for the reaction of S-2238 with thrombin.…”

Section: Results From Kinetic Studiesmentioning

confidence: 99%

“…The value of k 1 is modified by the ratio k 2 /(k −1 + k 2 ) = 101/(101+280) = 0.27 to 2.75 × 10 7 M −1 s −1 . (70–71) A crude estimate of the partitioning ratio of hemiketal formation over return to the enzyme-inhibitor complex plus proceeding to alkylation gives 115 for PPACK inhibition of thrombin. Judging from the rate constant for nucleophilic attack on S-2238 by Ser195 of thrombin, 101 s −1 at 25 °C, k h for hemiketal formation is probably < 100 s −1 .…”

Section: Results From Kinetic Studiesmentioning

confidence: 99%

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Proton Bridging in the Interactions of Thrombin with Small Inhibitors

et al. 2009

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Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human α-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant, ki/Ki, and the inhibition constant, Ki, for inhibition of human α-thrombin by PPACK are (1.1 ± 0.2) × 107 M−1 s−1 and (2.4 ± 1.3) × 10−8 M at pH 7.00 in 0.05 M phosphate buffer, 0.15 M NaCl, and 25.0 ± 0.1˚C, and in good agreement with previous reports. The activation parameters at pH 7.00, 0.05M phosphate buffer 0.15 M NaCl, are ΔH‡ = 10.6 ± 0.7 kcal/mol and ΔS‡ = 9 ± 2 cal/mol deg. The pH dependence of the second-order rate constants of inhibition is bell shaped. Values of pKa1 and pKa2 are 7.3 ± 0.2 and 8.8 ± 0.3, respectively, at 25.0 ± 0.1 °C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0, for pKa1 and 9.3 and 8.6 for pKa2. They inhibit thrombin over six orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 ± 0.1°C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors. But in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 °C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3–8.5. The peak at low field is an indication of the presence an SSHB at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 ± 0.2 (ϕ = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.

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Section: Results From Kinetic Studiesmentioning

confidence: 72%

Section: Results From Kinetic Studiesmentioning

confidence: 99%

Section: Results From Kinetic Studiesmentioning

confidence: 99%

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Proton Bridging in the Interactions of Thrombin with Small Inhibitors

et al. 2009

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“…We explain the inversion of product ratios by var- To test our hypothesis of variable T-domain loading, we have numerically fit reaction progress data with three simplified kinetic models (Scheme 1). 42,43 Progress curves were fit to these models using Dynafit. 44 The three models differ in the equations representing the initial catalytic steps of the NRPS.…”

Section: Resultsmentioning

confidence: 99%

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Stanišić

Hüsken

Stephan

et al. 2021

Preprint

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<div> <p>Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A- and C-domains determines the product specificity of NRPSs.</p> </div>

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“…The initial guesses were based on experimentally determined values of k cat , which were used for the estimation of k 2 ; the concentration of enzyme and substrate were determined independently. In a second round, k 1 , k −1 , k 2 and k 3 were optimized by fitting for individual runs of 6,000 and 4,000 fluorescence time data pairs, covering a total period of 6 and 4 sec, corresponding to experiments in absence and presence of heparin, respectively, on the same three-step mechanism mentioned above [25] . From the three-step reaction scheme, , , and were calculated values [24] .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2011

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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 (k 2 = 21±1 s−1) was much higher than the HNE deacylation step (k 3 = 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 k 1 2.4-fold and reducing k −1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k 2 value, whereas the k 3 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.

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Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with dynafit

Cited by 8 publications

References 30 publications

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

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

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