Crowding effects on EcoRV kinetics and binding

The cytosol of the cell contains high concentrations of small and large macromolecules, but experimental data are often obtained in dilute solutions that do not reflect in vivo conditions. We have studied the crowding effect that large macromolecules have on EcoRV cleavage by adding high-molecular-weight Ficoll 70 to reaction solutions. Results indicate that Ficoll has surprisingly little effect on overall EcoRV reaction velocity because of offsetting increases in V(max) and K(m), and stronger nonspecific binding. The changes in measured parameters can largely be attributed to the excluded volume effects on reactant activities and the slowing of protein diffusion. Covolume reduction upon binding appears to reinforce nonspecific binding strength, and k(cat) appears to be slowed by stronger nonspecific binding, which slows product release. The data also suggest that effective Ficoll particle volume decreases as its concentration increases above a few weight percent, which may be due to Ficoll interpenetration or compression.     

Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.     

Quantifying the effects of molecular orientation and length on two-dimensional receptor-ligand binding kinetics

Surface presentation of adhesion receptors influences cell adhesion, although the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the molecular orientation and length of adhesion receptors on the cell membrane affected two-dimensional kinetic rates of interactions with surface ligands. Interactions of P-selectin, E-selectin, and CD16A with their respective ligands or antibody were used to demonstrate such effects. Randomizing the orientation of the adhesion receptor or lowering its ligand- and antibody-binding domain above the cell membrane lowered two-dimensional affinities of the molecular interactions by reducing the forward rates but not the reverse rates. In contrast, the soluble antibody bound with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their orientation and length. These results demonstrate that the orientation and length of an adhesion receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding.     

Reflections on the kinetics of substrate binding

The principle questions which are still being asked about enzyme-substrate complex formation are examined. Data for the maximum rates of collision complex formation are reviewed. The limitations of past experimental procedures and the potentialities of new approaches are discussed together with methods that allow the distinction between collision complexes and subsequent events.     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.     

Transport and kinetics in sandwiched membrane bioreactors.

A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol. A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor. The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data.     

Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization

We describe here the selection of ultra-potent anti-respiratory syncytial virus (RSV) antibodies for preventing RSV infection. A large number of antibody variants derived from Synagis (palivizumab), an anti-RSV monoclonal antibody that targets RSV F protein, were generated by a directed evolution approach that allowed convenient manipulation of the binding kinetics. Palivizumab variants with about 100-fold slower dissociation rates or with fivefold faster association rates were identified and tested for their ability to neutralize virus in a microneutralization assay. Our data reveal a major differential effect of the association and dissociation rates on the RSV neutralization, particularly for intact antibodies wherein the association rate plays the predominant role. Furthermore, we found that antibody binding valence also plays a critical role in mediating the viral neutralization through a mechanism that is likely unrelated to antibody size or binding avidity. We applied an iterative mutagenesis approach, and thereafter were able to identify palivizumab Fab variants with up to 1500-fold improvement and palivizumab IgG variants with up to 44-fold improvement in the ability to neutralize RSV. These anti-RSV antibodies likely will offer great clinical potential for RSV immunoprophylaxis. In addition, our findings provide insights into engineering potent antibody therapeutics for other disease targets.     

Cyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics

To better understand the molecular mechanism of cAMP-induced and substrate-enhanced activation of type-I A-kinase, we measured the kinetics of A-kinase regulatory subunit interactions using a stopped-flow spectrofluorometric method. Specifically, we conjugated fluorescein maleimide (FM) to two separate single cysteine-substituted and truncated mutants of the type Ialpha regulatory subunit of A-kinase, RIalpha (91-244). One site of cysteine substitution and conjugation was at R92 and the other at R239. Although the emission from both conjugates changed with catalytic subunit binding, only the FM-R92C conjugate yielded unambiguous results in the presence of cAMP and was therefore used to assess whether a pseudosubstrate perturbed the rate of holoenzyme dissociation. We found that cAMP selectively accelerates the rate of dissociation of the RIalpha (91-244):C-subunit complex approximately 700-fold, resulting in an equilibrium dissociation constant of 130 nM. Furthermore, excess amounts of the pseudosubstrate inhibitor, PKI(5-24), had no effect on the rate of RIalpha (91-244):C-subunit complex dissociation. The results indicate that the limited ability of cAMP to induce holoenzyme dissociation reflects a greatly reduced but still significant regulatory catalytic subunit affinity in the presence of cAMP. Moreover, the ability of the substrate to facilitate cAMP-induced dissociation results from the mass action effect of excess substrate and not from direct substrate binding to holoenzyme.     

Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex

To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb. The results revealed that as the negative charge on the biosensor surface decreased, the binding kinetics and K(D) approached the accurate binding parameters more closely when measured in solution. Two potential causes for the artifactual Biacore surface-based measurements are (i) steric hindrance of antigen binding arising from an interaction of the negatively charged carboxymethyldextran matrix with the mAb, which is a highly basic protein with a pI of 9.4, and (ii) an electrostatic repulsion between the negatively charged antigen and the carboxymethyldextran matrix. Importantly, simple diagnostic tests can be performed early in the measurement process to identify these types of matrix-mediated artifacts.     

Heterogeneity of epidermal growth factor binding kinetics on individual cells.

Binding of fluorescein-conjugated epidermal growth factor (EGF) to individual A431 cells at 4 degrees C is measured by a quantitative fluorescence imaging technique. After background fluorescence and cell autofluorescence photobleaching corrections, the kinetic data are fit to simple models of one monovalent site and two independent monovalent sites, both of which include a first-order dye photobleaching process. Model simulations and the results from data analysis indicate that the one-monovalent-site model does not describe EGF binding kinetics at the single-cell level, whereas the two-site model is consistent with, but not proved by, the single-cell binding data. In addition, the kinetics of binding of fluorescein-EGF to different cells from the same coverslip often differ significantly from each other, indicating cell-to-cell variations in the binding properties of the EGF receptor.     

The kinetics of glucocorticoid binding to the soluble specific binding protein of mouse fibroblasts.

The kinetics of binding of glucocorticoids to the soluble, specific binding protein of mouse fibroblasts has been examined. The rate at which both potent and weak glucocorticoids achieve binding equilibrium is very slow. Second order rate constants of association range from 3 times 10-5 M- minus 1 min- minus 1 for cortisol to 6.7 times 10-5 M- minus 1 min- minus 1 for triamcinolone acetonide. Studies of the rates of binding at high steroid concentrations suggest that the slow rate of binding may be explained by a two-step mechanism. Active glucocorticoids, regardless of their potency, bind initially in a rapid manner to form a weak complex with the binding protein. The dissociation constant for the weak binding reaction is 0.87 times 10- minus 7 M for triamcinolone acetonide and 2.4 times 10- minus 7 M for cortisol. The weak binding complex becomes converted slowly to a tight complex. The first order rate constants for this conversion and the rate constants of dissociation from the tight complex have been determined for cortisol, dexamethasone and triamcinolone acetonide. The binding affinity of steroids of different biological potency is correlated with their rate of dissociation from this second tight binding state.     

Diffusional effects on the heterogeneous kinetics of two-substrate enzymic reactions.

When a two-substrate reaction is catalyzed by a surface bound enzyme, the diffusion of both substrates can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, limitations in substrate diffusion yield very different effects depending whether the two substrates have similar or different affinities for the enzyme. With two substrates of comparable affinities the diffusion of the two substrates can be limiting, and similar activity dependences on the two substrate concentrations are obtained. Under these conditions, diffusional limitations may only slightly influence half-maximal-activity substrate concentrations. With two substrates of widely different affinities, on the contrary, the rate of the enzymic reaction can only be limited by the diffusion of the high-affinity substrate, used at the lower concentration. Under these conditions, in the presence of diffusional limitations the activity dependence on the two substrate concentrations are highly different, and the half-maximal-activity concentration is increased and decreased for the high- and low-affinity substrates, respectively. The theoretical results are verified by experimental data previously obtained with collagen-bound aspartate aminotransferase and sorbitol dehydrogenase.     

Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.     

Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

Electrostatic effects on the kinetics of oxidation-reduction reactions of c-type cytochromes.

The kinetics of the oxidation-reduction reactions between horse heart cytochrome c, Euglena gracilis cytochrome c552, and ions (ascorbate, ferricyanide, and ferrocyanide) was investigated as a function of ionic strength at pH 7, 25 degrees C. The ionic strength was varied between 0.002 and 0.02 M. Data were analyzed according to four different functions of ionic strength. Results showed that the Kirkwood-Tanford smeared charge model holds well for the calculation of the activity coefficients and that the whole charges of these proteins are reflected in the rates of their reactions. Chemical modifications or changes in the pH that altered the charge of the proteins affected the primary salt effects as predicted by the smeared charge model.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair

MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (k(ON)=3 x 10(6) M(-1) s(-1)) and forms a stable complex with a half-life of 10 s (k(OFF)=0.07 s(-1)). When one or both nucleotide-binding sites on the MutS*mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5-7 s (k(OFF)=0.1-0.14 s(-1)), although MutS(ATP) becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS*mismatch complex forms rapidly (k(ON)=7.3 x 10(6) M(-1) s(-1)) and also dissociates rapidly, with a half-life of 0.4 s (k(OFF)=1.7 s(-1)). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATP-bound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.