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Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km.

ABSTRACT: If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.

SUBMITTER: Cornish-Bowden A

PROVIDER: S-EPMC1172593 | biostudies-other | 1976 Feb

REPOSITORIES: biostudies-other

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Estimation of rate and dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase.

Project description:Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD(+) in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pK(a) approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185-4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD(+) release from the enzyme-NAD(+)-ethanol product complex. This finding permits calculation of K(3), the dissociation constant for ethanol from the enzyme-NAD(+)-ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613-627). The calculations show that K(3) varies very little with pH in the range 5.95-8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme-NADH-acetaldehyde]/ [enzyme-NAD(+)-ethanol] at equilibrium. The values obtained were in the range 0.11+/-0.04, and this value together with estimates of K(3) was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.

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Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes.

Project description:The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein-nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd ) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA-based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. © 2018 by John Wiley & Sons, Inc.

| S-EPMC6391183 | biostudies-literature

Measuring dissociation rate constants of protein complexes through subunit exchange: experimental design and theoretical modeling.

Project description:Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off), provide structural and functional information on protein complexes. However, because all existing methods for measuring k(off) require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure k(off) using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that k(off) determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.

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Bovine ?-lactoglobulin is dimeric under imitative physiological conditions: dissociation equilibrium and rate constants over the pH range of 2.5-7.5.

Project description:The oligomerization of ?-lactoglobulin (?Lg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of ?Lg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for ?Lg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K(D)(2-1) fell close to or within the protein concentration range of ?5 to ?45 ?M, and at ?45 ?M the dimer predominated. No species larger than the dimer could be detected. The K(D)(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI?5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding ?Lg play a key role in the thermodynamics and kinetics of dimer association/dissociation.

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Acid dissociation constants of glycopeptides.

Project description:Glycopeptides containing mainly four amino acid residues in the sequence Asn-Leu-Thr-Ser, with small amounts of additional amino acid residues, were isolated from enzymic hydrolysates of hen's-egg albumin. Heterogeneity of the carbohydrate moiety was confirmed. Acid-base titrations showed that the alpha-amino group has a pKa value of 6.43 at 25 degrees C. The standard free engery and entropy changes associated with the ionization at 25 degrees C were 37.2kJ-mol-1 and -0.014kJ-mol-1- K-1 respectively. The complications arising in the interpretation of titration curves of the glycopeptides, which are heterogeneous with respect to the peptide chain, were considered and discussed in the light of the earlier suggestion that the titration curve of the glycopeptide might be interpreted as being due in part to a structure in which the hydroxyl group of the threonine residue is hydrogen-bonded to the beta-aspartamido oxygen atom [Neuberger & Marshall (1968) in Symposium on Foods - Carbohydrates and their Roles (Schultz, H.W., Cain, R.F. & Wrolstad, R.W., eds.), pp. 115-132, Avi Publishing Co., Westport, CT]. It is concluded that either the glycopeptides do not contain a hydrogen bond of that type, or, if they do, that it cannot be recognized by acid-base-titration studies.

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Project description:The isoelectric point is the pH at which a particular molecule is electrically neutral due to the equilibrium of positive and negative charges. In proteins and peptides, this depends on the dissociation constant (pKa) of charged groups of seven amino acids and NH+ and COO- groups at polypeptide termini. Information regarding isoelectric point and pKa is extensively used in two-dimensional gel electrophoresis (2D-PAGE), capillary isoelectric focusing (cIEF), crystallisation, and mass spectrometry. Therefore, there is a strong need for the in silico prediction of isoelectric point and pKa values. In this paper, I present Isoelectric Point Calculator 2.0 (IPC 2.0), a web server for the prediction of isoelectric points and pKa values using a mixture of deep learning and support vector regression models. The prediction accuracy (RMSD) of IPC 2.0 for proteins and peptides outperforms previous algorithms: 0.848 versus 0.868 and 0.222 versus 0.405, respectively. Moreover, the IPC 2.0 prediction of pKa using sequence information alone was better than the prediction from structure-based methods (0.576 versus 0.826) and a few folds faster. The IPC 2.0 webserver is freely available at www.ipc2-isoelectric-point.org.

| S-EPMC8262712 | biostudies-literature

Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.

Project description:Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd ). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.

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