Ligand Binding Steps Research Articles

Competition in drug binding and … the race to equilibrium.

Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some 10 years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants. Alike the velocity of product formation in enzymology, those fluxes refer to the velocity by which one target species converts into another one. Novel binding flux-based approaches are utilized to get a better visual insight into the "competition" between two drugs/ligands for a single target as well as between induced fit- and conformational selection pathways for a single ligand within a thermodynamic cycle. The present data were obtained by differential equation-based simulations. Early on, the ligand-binding steps "race" to equilibrium (i.e., when their forward and reverse fluxes are equal) at their individual pace. The overall/global equilibrium is only reached later on. For the competition association assays, this parting might produce a transient "overshoot" of one of the bound target species. A similar overshoot may also show up within a thermodynamic cycle and, at first glance, suggest that the induced fit pathway dominates. Yet, present findings show that under certain circumstances, it could rather be the other way round. Novel binding flux-based approaches offer visually attractive insights into crucial aspects of "complex" binding mechanisms under non-equilibrium conditions.

SAT-040 Structural Mechanisms of Parathyroid Hormone (PTH) Binding to Receptor and Regulation of Endosomal Signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR primarily activates the stimulatory G-protein, Gs, for adenylate cyclases that induce the production of cAMP. Our laboratory has demonstrated that PTH stimulates both transient and prolonged cAMP production at the plasma membrane and in endosomes, respectively. Activation of PKA through PTHR signaling in endosomes activates v-ATPases, which acidify the endosomal lumen. A decrease below pH 6.5 triggers the release of PTH from the receptor, thereby terminating Gs signaling. However, the structural mechanisms of PTH binding to PTHR and release from endosomal receptor are unclear. We studied the structural changes of PTH(1-34) upon binding to PTHR using nuclear magnetic resonance (NMR). We acquired 1H-15N HSQC, 2D 1H homonuclear NOESY, TOCSY, and 3D 15N-edited NOESY NMR spectra. The Hα chemical shifts of 15N-labeled PTH(1-34) residues, compared to “random coil” chemical shifts, demonstrate that at plasma membrane pH, unbound PTH consists of two halves: a helical C-terminal half (residues 18-34) and a less structured N-terminal half (1-13) connected by a random coil “hinge” (14-17). We hypothesize this hinge coordinates a two-step ligand binding mechanism, in which the helical C-terminal half of PTH binds to the extracellular domain of PTHR (ECD), followed by binding of the N-terminal half to the receptor transmembrane domain (TMD) and subsequent receptor activation. To investigate the first step of ligand binding, we compared the 1H-15N TROSY spectra of 15N-PTH(1-34) in the absence and presence of unlabeled ECD. HSQC peak intensities of PTH residues 21-34 are significantly reduced in the presence of ECD, suggesting that the flexibility of helical PTH(21-34) is compromised upon binding to ECD. Interestingly, peaks of L15 and S17, which participate in the hinge, disappear in the presence of ECD. In addition, new peaks for several PTH residues in both N- and C-terminal halves appear in the presence of ECD, indicating that ECD binding triggers new conformations of these residues. These data suggest that the increased rigidity of the hinge and new conformations of other N-terminal half residues position the N-terminal half for interaction with and binding to the TMD. To investigate the structural changes of PTH during endosomal acidification, we acquired HSQC spectra at pH 7.2, 6.5, and 5.9. These data reveal that the chemical shifts of PTH residues H9, H14, L15, and H32 are significantly affected by pH decrease, with H14 and L15 notably being most affected. Since H14 and L15 participate the hinge, we predict that conformational changes in the hinge as a result of pH decrease trigger ligand dissociation. Our work gives insight into the structural mechanisms of PTHR ligand binding and signaling regulation.

Structural mechanisms of parathyroid hormone (PTH) binding to receptor and regulation of endosomal signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR primarily activates the stimulatory G‐protein, Gs, for adenylate cyclases that induces the production of cAMP. Our laboratory has demonstrated that PTH stimulates both transient and prolonged cAMP production at the plasma membrane and in endosomes, respectively. Activation of PKA through PTHR signaling in endosomes activates v‐ATPases, which acidify the endosomal lumen. A decrease below pH 6.5 triggers the release of PTH from the receptor, thereby terminating Gs signaling. However, the structural mechanisms of PTH binding to PTHR and release from endosomal receptor are unclear. We studied the structural changes of PTH(1‐34) upon binding to PTHR using nuclear magnetic resonance (NMR). We acquired 1H‐15N HSQC, 2D 1H homonuclear NOESY, TOCSY, and 3D 15N‐edited NOESY NMR spectra. The H〈 chemical shifts of 15N‐labeled PTH(1‐34) residues, compared to “random coil” chemical shifts, demonstrate that at plasma membrane pH, unbound PTH consists of two halves: a helical C‐terminal half (residues 18–34) and a less structured N‐terminal half (1‐13) connected by a random coil “hinge” (14–17). We hypothesize this hinge coordinates a two‐step ligand binding mechanism, in which the helical C‐terminal half of PTH binds to the extracellular domain of PTHR (ECD), followed by binding of the N‐terminal half to the receptor transmembrane domain (TMD) and subsequent receptor activation. To investigate the first step of ligand binding, we compared the 1H‐15N TROSY spectra of 15N‐PTH(1‐34) in the absence and presence of unlabeled ECD. HSQC peak intensities of PTH residues 21‐34 are significantly reduced in the presence of ECD, suggesting that the flexibility of helical PTH(21‐34) is compromised upon binding to ECD. Interestingly, peaks of L15 and S17, which participate in the hinge, disappear in the presence of ECD. In addition, new peaks for several PTH residues in both N‐ and C‐terminal halves appear in the presence of ECD, indicating that ECD binding triggers new conformations of these residues. These data suggest that the increased rigidity of the hinge and new conformations of other N‐terminal half residues position the N‐terminal half for interaction with and binding to the TMD. To investigate the structural changes of PTH during endosomal acidification, we acquired HSQC spectra at pH 7.2, 6.5, and 5.9. These data reveal that the chemical shifts of PTH residues H9, H14, L15, and H32 are significantly affected by pH decrease, with H14 and L15 notably being most affected. Since H14 and L15 participate in the hinge, we predict that conformational changes in the hinge as a result of pH decrease trigger ligand dissociation. Our work gives insight into the structural mechanisms of PTHR ligand binding and signaling regulation.Support or Funding InformationNIH R01 DK116780 and DK102495This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimizing hybrid LC-MS/MS binding conditions is critical: impact of biotransformation on quantification of trastuzumab.

Background: Hybrid ligand-binding (LB) LC-MS/MS protein quantitative assays involve a LB step for analyte enrichment that has less stringent requirements than the conventional LB assays. Results: Herceptin™(trastuzumab) binding to HER2 extracellular domain was evaluated using on-bead and off-bead capture formats. The two formats yielded significantly different trastuzumab concentrations in human and monkey serum pharmacokinetic samples. Biotransformations, including deamidation of asparagine and isomerization of aspartic acid near the complementarity-determining regions of trastuzumab, had a profound impact on the LB step for analyte enrichment and trastuzumab quantification. Conclusion: Quantitative measurements were profoundly impacted by LB conditions in a hybrid LB LC-MS/MS protein assay due to biotransformations. Therefore, similar to conventional LB assays, binding conditions should be carefully evaluated during assay development.

Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase.

Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein-ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.

Single Molecule Ligand Binding FRET at HCN2 Channel Domains in Zero-Mode Waveguides

Ligand binding is a critical component of many signaling pathways. Our understanding of the kinetic and mechanistic steps involved in the components comprising these pathways has been hampered by the difficulty of observing the ligand binding step itself, rather than some downstream measure of activity (e.g. current through an ion channel). Here, we use FRET to directly observe individual binding events of a fluorescently tagged cyclic nucleotide (fcAMP; Kusch et al. 2010) to its binding domain in hyperpolarization activated cyclic nucleotide gated (HCN2) channels at single molecules. To resolve single binding events at physiological micromolar concentrations of ligand, binding domains were tethered in zero-mode waveguides to reduce background fluorescence from unbound ligands (Levene et al., 2003). Our observations directly reveal the ligand binding process of fcAMP to its recognition site in HCN2 channels.

Theory for rates, equilibrium constants, and Brønsted slopes in F1-ATPase single molecule imaging experiments

A theoretical model of elastically coupled reactions is proposed for single molecule imaging and rotor manipulation experiments on F1-ATPase. Stalling experiments are considered in which rates of individual ligand binding, ligand release, and chemical reaction steps have an exponential dependence on rotor angle. These data are treated in terms of the effect of thermodynamic driving forces on reaction rates, and lead to equations relating rate constants and free energies to the stalling angle. These relations, in turn, are modeled using a formalism originally developed to treat electron and other transfer reactions. During stalling the free energy profile of the enzymatic steps is altered by a work term due to elastic structural twisting. Using biochemical and single molecule data, the dependence of the rate constant and equilibrium constant on the stall angle, as well as the Børnsted slope are predicted and compared with experiment. Reasonable agreement is found with stalling experiments for ATP and GTP binding. The model can be applied to other torque-generating steps of reversible ligand binding, such as ADP and Pi release, when sufficient data become available.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2]4+, which consists of two Ru(phen)2dppz2+ moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

Monitoring the Work of a Single Subunit in Homotetrameric CNGA2 Channels

Cyclic nucleotide-gated (CNG) channels mediate signal transduction in photoreceptors and olfactory cells. Wild-type olfactory CNG channels are composed of three types of subunits, CNGA2, CNGA4, and CNGB1b. Out of these only CNGA2 subunits can form functional homotetrameric channels when expressed in heterologous systems. Homotetrameric CNGA2 channels are very useful for studying elementary biophysical processes. However, even for homotetrameric channels the transmission of the ligand binding to the pore opening is still a mystery. Undoubtedly clear is only that the activation of the channels is not generated by independently operating but by interacting subunits. We are interested in how a single subunit embedded in a channel binds a ligand and evokes channel activation, i.e. for the whole channel we intend to kinetically dissect the molecular gating mechanism induced by the first ligand binding step. We constructed tetrameric concatamers of CNGA2 channels with various numbers of wild-type and mutated binding domains of high and extremely low affinity. Ligand binding was measured by confocal patch-clamp fluorometry (frame rate up to 277 images per second) using a fluorescent cGMP analogue. Our results show that a single subunit operates a channel only at 23-fold higher ligand concentrations than a tetrameric channel composed of four cooperating subunits. Nevertheless, the single subunit opens the channel pore to the full conductance level. Moreover, we show that Markovian models with a discrete number of states surprisingly fail to describe the action of the only wild-type subunit. Instead, a model consisting of three separate but intimately coupled processes proved to be adequate for describing channel activation, containing a rapid ligand binding close to the diffusion limit, a continuous conformational diffusion upon gating consisting of two components, and a rapid and discrete pore action.

Thermodynamic aspects of cAMP dependent protein kinase catalytic subunit allostery.

Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50°C). The thermodynamic parameters ΔH and ΔS for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (ΔH). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.

Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain

The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson–Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine-scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures. Proteins 2013; 81:1931–1943. © 2013 The Authors. Proteins published by Wiley Periodicals, Inc.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

Plasticity of Cytochrome P450 2B4 as Investigated by Hydrogen-Deuterium Exchange Mass Spectrometry and X-ray Crystallography

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.

Characterization of the Tritium-Labeled Analog of L-threo-β-Benzyloxyaspartate Binding to Glutamate Transporters

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo-beta-benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([3H]ETB-TBOA). [3H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na+-dependence of [3H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na+, suggesting that Na+ binding in the ligand binding step is different from Na+ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [3H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

Regions of Drosophila Notch That Contribute to Ligand Binding and the Modulatory Influence of Fringe

Two glycosyltransferases that transfer sugars to epidermal growth factor (EGF) domains, OFUT1 and Fringe, regulate Notch signaling. To characterize the impact of glycosylation at the 23 consensus O-fucose sites in Drosophila Notch, we conducted deletion mapping and site-specific mutagenesis and then assayed the binding of soluble forms of Notch to cell-surface ligands. Our results support the conclusion that EGF11 and EGF12 are essential for ligand binding, but indicate that other EGF domains also make substantial contributions to ligand binding. Characterization of Notch deletion constructs and O-fucose site mutants further revealed that no single site or region can account for the influence of Fringe on Notch-ligand binding. Additionally, we observed an influence of Fringe on a Notch fragment including only 4 of its 36 EGF domains (EGF10-13). Together, our observations imply that glycosylation influences Notch-ligand interactions through a distributive mechanism that involves local interactions with multiple EGF domains and led us to suggest a structural model for how Notch interacts with its ligands.

Kinetic evidence for a substrate-induced fit in phosphonoacetaldehyde hydrolase catalysis.

Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4-dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK(a) of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover.

Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity.

The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.

Investigation of the α1-Glycine Receptor Channel-Opening Kinetics in the Submillisecond Time Domain

The activation and desensitization kinetics of the human α 1-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100- μs time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and β-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k +1 ∼10 7 M −1 s −1, and dissociation, k −1 = 1900 s −1; 2) the rates of receptor-channel opening, k op = 2200 s −1, and closing, k cl = 38 s −1; 3) the receptor desensitization rate, α = 0.45 s −1; 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n ≥ 3; and 5) the maximum receptor-channel open probability, p 0 > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.

Protonic equilibria in the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase.

Oxidation of thioester substrates in the medium-chain acyl-CoA dehydrogenase involves alpha-proton abstraction by the catalytic base, Glu376, with transfer of a beta-hydride equivalent to the flavin prosthetic group. Polarization of bound acyl-CoA derivatives by the recombinant human liver enzyme has been studied with 4-thia-trans-2-enoyl-CoA analogues. Polarization is maximal at low pH, with an apparent pK of 9.2 for complexes with the C8 analogue, and progressively lower pK values as the length of the chain increases. This pH effect reflects ionization of the catalytic base, since polarization of a variety of enoyl-CoA analogues by the Glu376Gln mutant is pH independent. Binding of these ligands is accompanied by uptake of about 1 proton with the wild-type enzyme, but only about 0.1 proton with the Glu376Gln mutant. Rapid reaction studies show that proton uptake with the wild-type enzyme occurs at the same rate as polarization of the enoyl-CoA thioester, but is much slower than the initial ligand binding step. Studies with 6-OH-FAD-substituted enzyme show that this isomerization reaction also influences the flavin prosthetic group inducing deprotonation to the green anionic form. The failure of the bound thioether analogue, octyl-SCoA, to elicit pK shifts to flavin and Glu376 shows the importance of the thioester carbonyl oxygen in modulating key properties of the medium-chain enzyme. The role of thioester-mediated desolvation within the active site of the acyl-CoA dehydrogenases is discussed.