Kinetics for the Life Sciences

Gutfreund, H.

doi:10.1017/cbo9780511626203

Cited by 275 publications

(198 citation statements)

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“…We used transition state theory and the temperature-dependence of the rate of phospho-cysteine formation with the substrate pNPP to shed further light on the kinetic and physical mechanism of Cdc25B (Gutfreund 1995). The thermodynamic transition state activation parameters ΔH ‡ and TΔS ‡ for the rate of chemistry (k 2 ) at standard conditions were determined with the Eyring plot using Equation 2.…”

Section: Resultsmentioning

confidence: 99%

“…The moderate magnitude of the activation enthalpy for Cdc25B association with protein substrate hints at the significant but not exclusive role of free diffusion in the association of protein substrate. A strictly diffusion controlled process is expected to have a low activation enthalpy of ~ 4 kcal/mol, whereas if the association is largely controlled by internal factors such as massive residue rearrangements, the ΔH ‡ tends to be greater than 20 kcal/mol (Gutfreund 1995). The significant favorable entropic contribution of 4 kcal/mol is consistent with the desolvation that must occur during formation of the large protein-protein interface between Cdc25B and its protein substrate (Sohn and others 2005).…”

Section: The Temperature Dependence Of Es Formation For Cdc25b With mentioning

confidence: 99%

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Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Sohn

Rudolph

2007

Biophysical Chemistry

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Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (ΔH ‡ of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298K. Phosphate transfer from Cdk2-pTpY/CycA to enzyme (ΔH ‡ of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (ΔH ‡ of 18 kcal/mol), yet entropically less favorable (TΔS ‡ of 2 vs. -6 kcal/mol at 298K, respectively). By measuring the temperature-dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy-entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme -substrate association involves tight and specific contacts at the remote docking site and that phosphotransfer from Cdk2-pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.

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Section: Resultsmentioning

confidence: 99%

Section: The Temperature Dependence Of Es Formation For Cdc25b With mentioning

confidence: 99%

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Sohn

Rudolph

2007

Biophysical Chemistry

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“…Binding of a peptide derived from hirudin (Hir [52][53][54][55][56][57][58][59][60][61][62][63][64][65] ) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S 2 and S 3 to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P 2 and P 3 .…”

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confidence: 99%

The Role of Glu192 in the Allosteric Control of the S2′ and S3′ Subsites of Thrombin

Marque¹,

Spuntarelli²,

Juliano³

et al. 2000

Journal of Biological Chemistry

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Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir 52-65 ) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S 2 and S 3 to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P 2 and P 3 . Regardless of the amino acids, Hir 52-65 decreased, and heparin increased the k cat /K m value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu 192 participates in this modulation. We have examined the role of Glu 192 by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P 2 residue was cleaved with a k cat /K m value only 49 times higher than the one having the "least favorable" P 2 residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P 3 residues and its strong aversion for negatively charged P 3 residues. Furthermore, both Hir 52-65 and heparin increased the k cat /K m value of substrate hydrolysis. We conclude that Glu 192 is critical for the P 2 and P 3 specificities of thrombin and for the allostery mediated through exosite 1.Thrombin (1, 2) is a multifunction serine protease finely tuned through: 1) restrictions fulfilled by the nature of the P 3 to P 3 Ј residues of the substrate, 1 that the S 3 to S 3 Ј subsites of the protease must accommodate, 2) steric hindrance resulting from surface loops, that limit access to the active site, 3) secondary exosites (one apolar and two positively charged) that strengthen the binding of cognate macromolecules (e.g. substrates such as fibrinogen but also inhibitors such as hirudin), and 4) allosteric control conferred by various cofactors (3). Several lines of evidence suggest that Glu 192 is a major player in the specificity of thrombin (4).2 Overall, hydrolysis by thrombin of its "best" substrates is little affected by the E192Q mutation, but new activities emerge that are severely restrained in normal thrombin. The E192Q mutant activates bovine factor X (5) and is efficiently inactivated by the serpin ␣ 1 -antitrypsin (6) and by the bovine pancreatic trypsin inhibitor (BPTI) 3 (7, 8). Glu 192 also seems to participate in the allosteric modulation of thrombin (4, 9). E192Q activates the anticoagulant protein C faster than thrombin; however, in the presence of the cofactor thrombomodulin, this difference disappears (4). Notably, the side chain of Glu 192 changes its conformation upon binding of a ligand to exosite 1 (10 -12).Among the effectors altering thrombin catalysis, molecules as diverse as inorganic ions, tryptamine analogs, polysaccharides, and proteins (or peptides derived from these proteins) have been identified. Sodium ions are allosteric modulators of thrombin, along with other serine proteases having a tyrosine in position...

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“…Any proposed model for the progress of an enzyme reaction and the effects of different concentrations of substrates, inhibitors, activators, as well as changes in many other conditions can be described by a set of differential equations (10). The nonlinear fitting programs referred to above can be fed with these equations, and the data can be fitted to the model.…”

Section: From Tabulation To Plottingmentioning

confidence: 99%

Numerical methods and computing in laboratories: From log tables and slide rules to laptop computers during a lifetime

Gutfreund

2010

IUBMB Life

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Kinetics for the Life Sciences

Cited by 275 publications

References 0 publications

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

The Role of Glu192 in the Allosteric Control of the S2′ and S3′ Subsites of Thrombin

Numerical methods and computing in laboratories: From log tables and slide rules to laptop computers during a lifetime

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