Negative Homotropic Cooperativity Research Articles

Negative Homotropic Cooperativity in Guest Binding of a Trisporphyrin Double Cleft.

Invited for the cover of this issue is the group of Takeharu Haino at Hiroshima University. The image depicts the host-guest complex of a trisporphyrin double cleft with an electron-deficient aromatic molecule, which shows negative cooperativity in guest binding. Read the full text of the article at 10.1002/chem.202300107.

Front Cover: Negative Homotropic Cooperativity in Guest Binding of a Trisporphyrin Double Cleft (Chem. Eur. J. 32/2023)

A triple-layered trisporphyrin molecule possessing two cleft cavities showed negative homotropic cooperativity in the host–guest complex. When an electron-deficient guest molecule (yellow) was accommodated within one of the cleft cavities, the electronic structure of the porphyrin was altered, and the binding power of the remaining cavity was reduced. More information can be found in the Research Article by T. Haino and co-workers (DOI: 10.1002/chem.202300107).

Negative Homotropic Cooperativity in Guest Binding of a Trisporphyrin Double Cleft.

Thenegative homotropic allostery of a triple-layered trisporphyrin cleft with two guest binding sites was confirmed. The X-ray crystal structures of the 1:2 host-guest complexes showed that the trisporphyrin accommodated two guest molecules within the cleft via π-π stacking and donor-acceptor interactions. In solution,1H NMR and Job plots showed 1:2 host-guest complexes. Isothermal titration calorimetry (ITC) and UV/vis absorption spectroscopy were employed to evaluate the binding constants and cooperativities. The guest binding of the trisporphyrin showed negative cooperativity and noncooperativity depending on the structures of the guest molecules. The correlations between the interaction parameters (α) and Hill constants were determined. ITC experiments showed that the host-guest complexation of trisporphyrin with electron-deficient guests incurred an enthalpy penalty in the successive guest binding process. DFT calculations revealed that the first guest binding reduced the electron density of the central porphyrin plane, which led to an energetic penalty that weakened the successive binding process.

Energetics of Allosteric Negative Coupling in the Zinc Sensor S. aureus CzrA

The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn(2+). Zn(2+) binds to the two identical binding sites on the free CzrA homodimer in two discernible steps. A larger entropic driving force Delta(-TDeltaS) of -4.7 kcal mol(-1) and a more negative DeltaC(p) characterize the binding of the first Zn(2+) relative to the second. These features suggest a modest structural transition in forming the Zn(1) state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn(2+) binding (Delta(DeltaG) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn(2+) binding to the CzrA*CzrO complex (Delta(DeltaG) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(DeltaH) and Delta(-TDeltaS) values both small and slightly positive. In short, Zn(2+) binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (DeltaG(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrA*CzrO reference states (DeltaH(c)(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn(2) state off the DNA (TDeltaS(c)(t) = -3.1 kcal mol(-1), i.e., DeltaS(c)(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.

Cooperative Binding of Acetaminophen and Caffeine within the P450 3A4 Active Site

Acetaminophen (N-acetyl-p-aminophenol, APAP) is a commonly used analgesic/antipyretic. When oxidized by P450, a toxic APAP metabolite is generated. Human P450 3A4 was expressed in Escherichia coli , purified, and reconstituted using artificial liposomes. Oxidation of APAP by P450 3A4, as detected by the formation of its glutathione adduct, was found to exhibit negative homotropic cooperativity with a Hill coefficient of 0.7. In the presence of caffeine, the observed kinetics were close to classical Michaelis-Menten kinetics with a Hill coefficient approaching 1. In order to probe for a potential repositioning of APAP within the P450 3A4 pocket in the presence of caffeine, NMR T1 paramagnetic relaxation techniques were used to calculate distances from the P450 3A4 heme iron to protons of APAP alone and in the presence of caffeine. Both APAP and caffeine were found to bind at the active site in proximity to the heme iron. When APAP was incubated with P450 3A4, the acetamido group of APAP was found to be closest to the heme iron consistent with the amide group of APAP weakly associating with the heme iron. The addition of caffeine disrupted the ability of APAP to coordinate with the heme iron of P450 3A4 and enhanced the rate of oxidation to its toxic metabolite.

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.

Structural Insights into Homo- and Heterotropic Allosteric Coupling in the Zinc Sensor S. aureus CzrA from Covalently Fused Dimers

The Zn(II)/Co(II)-sensing transcriptional repressor, Staphylococcus aureus CzrA, is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N(3)O metal sensor coordination sites. A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins. Here, we exploit covalent (Gly(4)Ser)(n)() linkers of variable length in "fused" CzrAs, where n = 1 (designated 5L-fCzrA), 2 (10L-fCzrA), or 3 (15L-fCzrA), as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees. While 15L-fCzrA exhibits properties most like homodimeric CzrA, shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly. Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity. However, Zn(II) negatively regulates DNA binding of all fCzrAs, with allosteric coupling free energies (DeltaG(1)(c)) of 4.6, 3.1, and 2.7 kcal mol(-1) for 15L-, 10L-, and 5L-fCzrAs, respectively. Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in DeltaG(1)(c) = 2.6 kcal mol(-1) or approximately 83% that of 10L-fCzrA; in contrast, homodimeric H97N CzrA gives DeltaG(1)(c) = 0. (1)H-(15)N HSQC spectra acquired for wt-, 10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted. Thus, occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding.

NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity

Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.

Positive Heterotropic Cooperativity for Selective Guest Binding via Electronic Communications through a Fused Zinc Porphyrin Array

Upon binding with C60 and diamines, such as 4,4'-bipyridine (bpy) and N,N,N',N'-tetramethylhexane-1,6-diamine (TMHDA), cyclic host 1 possessing two electronically coupled binding sites displays negative homotropic cooperativity and positive heterotropic cooperativity, and their ternary mixtures preferentially form inclusion complexes with hetero-guest pairs 1 supersetC60*bpy and 1 supersetC60*TMHDA under appropriate conditions. Spectroscopic titration profiles in toluene at 20 degrees C demonstrated that the association constants (Kassoc) of C60 with monodiamine complexes 1 supersetbpy (2.8 x 105 M-1) and 1 supersetTMHDA (1.5 x 105 M-1) are 8.5 and 4.5 times greater than that of C60 with guest-free 1 (3.3 x 104 M-1), respectively. On the other hand, mono-C60 complex 1 supersetC60 was 6.1 times more accessible than guest-free 1 toward TMHDA. Absorption spectroscopy in the absence of 1 indicated no direct interaction between C60 and diamines.

Is There a Toxicological Advantage for Non-hyperbolic Kinetics in Cytochrome P450 Catalysis?: FUNCTIONAL ALLOSTERY FROM “DISTRIBUTIVE CATALYSIS”

The cytochrome P450s (CYPs) are the major enzymatic detoxification and drug metabolism system. Recently, it has become clear that several CYP isoforms exhibit positive and negative homotropic cooperativity. However, the toxicological implications of allosteric kinetics have not been considered, nor understood. The allosteric kinetics are particularly enigmatic in several respects. In many cases, CYPs bioactivate substrates to more toxic products, thus making it difficult to rationalize a functional advantage for positive cooperativity. Also, CYPs exhibit cooperativity with many structurally diverse ligands, in marked contrast to the specificity observed with other allosteric systems. Here, kinetic simulations are used to compare the probabilistic time- and concentration-dependent integrated toxicity function during conversion of substrate to product for CYP models exhibiting Michaelis-Menten (non-cooperative) kinetics, positive cooperativity, or negative cooperativity. The results demonstrate that, at low substrate concentrations, the slower substrate turnover afforded by cooperative CYPs compared with Michaelis-Menten enzymes can be a significant toxicological advantage, when toxic thresholds exist. When present, the advantage results from enhanced "distribution" of toxin in two pools, substrate and product, for an extended period, thus minimizing the chance that either exceeds its toxic threshold. At intermediate concentrations, the allosteric kinetics can be a modest advantage or modest disadvantage, depending on the kinetic parameters. However, at high substrate concentrations associated with a high probability of toxicity, fast turnover is desirable, and this advantage is provided also by the cooperative enzymes. For the positive homotropic cooperativity, the allosteric kinetics minimize the probability of toxicity over the widest range of system parameters. Furthermore, this apparent functional cooperativity is achieved without specific molecular recognition that is the hallmark of "traditional" allostery.

Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.

Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.

Negative homotropic cooperativity in rat muscle AMP deaminase: A kinetic study of the inhibition of the enzyme by ATP

1. 1.|Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. 2.|Up to 10 μM, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. 3.|AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)—AMP deaminase complex stabilized by NaBH 4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. 4.|A plausible explanation of the observed cooperativety is that ATP induces different conformation states of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. 5.|Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.

Receptor binding of glucagon and adenosine 3′,5′-monophosphate accumulation in isolated rat fat cells

• The binding of glucagon and its effect on adenosine 3′,5′-monophosphate (cyclic AMP) accumulation and glycerol release was assessed in isolated rat fat cells at 37°C. • The dissociation constant of the displaceable (saturable, specific) binding was about 1.5 nM. The rate constant of dissociation ( k −1) varied from 8 8.6 · 10 −4 s −1 to 3.3 · 10 −3 s −1, i.e. T 1 2 varied from 800 s to 200 s. Within an experiment, k −1 was the same when cells with prebound 125I-labelled glucagon were suspended in medium containing either no glucagon or 1 μM of unlabelled glucagon. Negative homotropic cooperativity therefore seemed absent. The half-time of association of 70 pM 125I-labelled glucagon was of the same order of magnitude as that of dissociation. • The conversion of prelabelled ATP to labelled cyclic AMP was enhanced by glucagon with a time lag of less than 20 s. The maximal accumulation was reached at 2–5 min both in the absence and in the presence of 2.5 mM theophylline. The concentration of glucagon causing half-maximal accumulation of cyclic AMP at 5 min was about 2 nM both in the absence and in the presence of theophylline. Glucagon stimulated glycerol release half-maximally at a concentration of about 2 nM in 1 h incubations. • The des-His 1-glucagon exhibited about half of the maximal effect of glucagon on cyclic AMP accumulation even though it was able to inhibit binding of 125I-labelled glucagon to the same extent as glucagon. Half-maximal displacement and half-maximal effect were obtained with about 200 nM of des-His 1-glucagon. • The results are compatible with the following model for the action of glucagon on adipocytes. The adenylyl cyclase is stimulated in a dose-dependent fashion at the low receptor occupancies obtained 20 s after the addition of glucagon in submaximally stimulating concentrations. The increase in cyclic AMP concentration is antagonized so that the maximal accumulation with a given concentration of glucagon is obtained after a few minutes even though the receptor occupancy continues to increase. The histidine residue is necessary for maximal activation of the adenylyl cyclase.

Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+. Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested.

Transport of sugars and amino acids in bacteria. XVI. Theory and evaluation of a model for the membrane transport reaction mediated by a single carrier with three binding sites for substrate.

A novel model is presented for bacterial active transport reactions which show a curvilinear Eadie-Hofstee plot and negative homotropic cooperativity in the kinetics of substrate uptake. Various models of a single carrier with multi-binding sites for substrate were constructed and examined theoretically. The fit of these models with experimental data on the kinetics of branched chain amino acid transport reactions were tested by iterative computation using the non-linear least square method. The transport model which fitted the experimental data best consisted of a single carrier with three binding sites for substrate in which one of the substrate-carrier complexes, CSS, is not active in translocating substrate across the cytoplasmic membrane. The mechanism of homeostatic regulation of the intracellular concentration of amino acids by active transport systems is discussed on the basis of this transport model.

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K m values were determined, these were K m H, 2·9 × 10 −4 M and K m L, 4·3 × 10 −5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.

Deoxycytidine Kinase: III. KINETICS AND ALLOSTERIC REGULATION OF THE CALF THYMUS ENZYME

Abstract The kinetic behavior of calf thymus deoxycytidine kinase was studied over a 100-fold variation in deoxycytidine concentration, and several abrupt transitions were observed in slopes of double reciprocal plots, suggesting negative homotropic cooperativity in the binding of this substrate. This is in apparent contrast to the simpler kinetics seen when an alternate substrate, deoxyguanosine, or the substrate analogue, 1-β-d-arabinofuranozylcytosine, was used in place of deoxycytidine. Convex reciprocal plots were also obtained with variable ATP-Mg2- or with an alternative phosphate donor, dTTP-Mg2-, although the latter yielded more marked negative curvature and lower Vmax values. Inhibition by end product, dCTP, was complex, particularly when deoxycytidine was varied over a wide range of concentrations, but below the first transition concentration dCTP plotted as a noncompetitive inhibitor. With variable ATP-Mg2-, dCTP gave the appearance of noncompetitive inhibition at low ATP-Mg2-, but shifted to apparent competitive inhibition against high phosphate donor concentrations. End product inhibition is overcome by dTTP at low deoxycytidine concentrations, but with increased nucleoside concentrations dTTP replaces the dCTP effect with its own highly complex pattern of inhibition. Like dTTP, UDP also stimulates at low nucleoside concentrations and becomes inhibitory as the deoxycytidine concentration is elevated. However, this inhibition can be reversed by dTTP, provided that the ATP-Mg2- concentration is limiting.