Isomerization Constant Research Articles

Application of the Matrix Method for Calculating Internal Equilibrium Constants and Complex Formation Microconstants

This review is devoted to the matrix method of simulating complex formation in solution. The applicability of the method to the interpretation of spectrometric and calorimetric data for systems containing simultaneously a large number of complex forms is shown. The works where this method was used to calculate the internal equilibrium constants and cis/trans isomerization constants were analyzed. Using the matrix simulation method, the cooperativity of ammonia binding by Mg2+, Ca2+, Zn2+, Cd2+, and Ni2+ ions in aqueous solution was demonstrated. For halide complexes of Pt(II), Pd(II) and Au(III) with a square-planar structure, the difference between the mutual influence of ligands in the cis and trans isomers is shown. The question of verifying the hypothesis about the geometry of the coordination polyhedron of Cu2+ compounds formed in the processes of equilibrium stepwise complex formation in mixed solvents is considered.

Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria

We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic receptors in myocardial homogenates as measured in competitive binding experiments with [3H]N‐methylscopolamine. This subset of muscarinic binding sites is thought to represent a receptor‐Gi/o protein complex. We measured agonist binding to this site directly using the agonist radioligand, [3H]oxotremorine‐M, and completed agonist/[3H]oxotremorine‐M competitive binding experiments to estimate ligand affinity for this site in the guinea pig myocardium. We estimated a log affinity constant of approximately 8.2 for oxotremorine‐M in competitive binding experiments with [3H]oxotremorine‐M in the presence of 140 mM Na+. The allosteric agonist, LY211,9620 caused about a twofold increase in the affinity of oxotremorine‐for this site. In the presence of low sodium, LY211,9620 had little effect on oxotremorine‐M affinity, suggesting that in presence of both LY211,9620 and low sodium, the observed affinity of oxotremorine‐M is nearly the same as the active state affinity. Under these conditions, the log affinity constant of oxotremorine‐M was approximately 8.8. This value was similar to that estimated in functional studies on the atria (log Kact, 9.1 ± 0.10). These functional studies also provided estimates of the log Kact and Kinact values of the allosteric agonist LY211,9620 (6.3 and 5.1, respectively), the log isomerization constant of the unoccupied M2 muscarinic receptor (Kq, −3.18 ± 0.11), and the log sensitivity constant of the signaling pathway (log KE, 1.87 ± 0.27). Receptor‐state affinity constants provide a more powerful approach to structure activity relationships and to the measurement of agonist bias.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estimation of the receptor-state affinity constants of ligands in functional studies using wild type and constitutively active mutant receptors: Implications for estimation of agonist bias

We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.

Analysis of ligand bias in functional studies involving the allosteric modulation of G protein‐coupled receptors (654.6)

The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its ability to activate downstream signaling events. In this report, we describe a reverse‐engineering strategy for estimating these microscopic constants. Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein‐coupled receptor under conditions of partial receptor inactivation and allosteric modulation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands ‐ including inverse agonists ‐ and the efficacy of the unoccupied receptor (i.e., constitutive activity). We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor‐signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe‐dependent allosteric interactions and the selectivity of agonists for different signaling pathways.Grant Funding Source: NIH grant GM 69829

Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: Implications for ligand bias

IntroductionThe affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. MethodsOur approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands – including inverse agonists – and the efficacy of the unoccupied receptor (i.e., constitutive activity). Results and discussionWe validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration–response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor.

Calculations of microconstants and equilibrium formation constants for platinum(II) and palladium(II) halide complexes in solution

Distribution diagrams and formation functions for halide complexes [M(H2O)4 − nCln]2 − n (M = Pt(II) or Pd(II)) and [PdCl4 − nBrn]2− (n = 0–4) in solution are analyzed in terms of the matrix model. Equilibrium constants for binding the first ligand \(\left( {\bar K} \right)\) and corrections for the mutual influence between ligands (ω) in the course of complex formation in solution are calculated. In examples analyzed, the substitution of chloride ion for water in the coordination sphere of platinum(II) and palladium(II) is an anti-cooperative process. The substitution of bromide ion for chloride ion in the coordination sphere of [PdCl4]2− is weakly cooperative. Quantum-chemical calculations show that platinum(II) and palladium(II) cis-bisaquadichloro complexes in the gas phase are thermodynamically less stable than trans-isomers. The cis-trans isomerization constants in the gas phase calculated by the DFT method and those found for solutions using the matrix model have the same order of magnitude.

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (Kisom) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated Kisom values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway.As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the Kisom calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.

An Enzyme-linked Receptor Mechanism for Nitric Oxide-activated Guanylyl Cyclase

Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents, such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides, and allosteric agents bind and the protein undergoes a conformational change, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all of the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all of the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism enhances understanding of the behavior of the receptor in a cellular setting.

Structure–Activity Relationships Among the Nitrogen Containing Bisphosphonates in Clinical Use and Other Analogues: Time-Dependent Inhibition of Human Farnesyl Pyrophosphate Synthase

The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.

13C NMR studies of isomerization of D-glucose in an aqueous solution of Ca(OH)2. The effect of molecular oxygen

Isomerization of D-glucose to fructose and mannose in aqueous solutions of Ca(OH)2 with the initial pH 11.4 in a temperature interval of 20–90 °C was studied by 13C NMR spectroscopy in the presence and absence of dissolved oxygen. In the presence of oxygen, the apparent equilibrium isomerization constant is much lower than that in the absence of oxygen. This is related to the oxidation of monosaccharides to formic and aldonic acids, a decrease in the pH of solutions, and cessation of isomerization at pH < 9.

Interaction of the 20 kDa and 63 kDa Fragments of Anthrax Protective Antigen: Kinetics and Thermodynamics

The action of anthrax toxin begins when the protective antigen (PA(83), 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA(20) (20 kDa) and PA(63) (63 kDa). After PA(20) dissociates, receptor-bound PA(63) spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol. Treatment of PA(83) with trypsin yielded PA(63) and a form of PA(20) lacking unstructured regions at the N- and C-termini. We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution. We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, k(off), of 3.3 x 10(-)(2) s(-)(1) (t(1/2) of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (k(on) = 1.4 x 10(5) M(-)(1) s(-)(1)) was followed by a slower unimolecular step (k = 3.5 x 10(-)(3) s(-)(1)) with an equilibrium isomerization constant, K(iso), of 2.1. The two-step mechanism most consistent with the data is one in which the dissociation of the PA(20).PA(63) complex is followed by an isomerization in the PA(63) moiety. Our results indicate that, following the cleavage of PA on the cell surface, PA(20) is largely dissociated within a minute. A slow isomerization step in PA(63) may then potentiate it for oligomerization and subsequent steps in toxin action.

A homotropic two-state model and auto-antagonism

BackgroundBell-shaped and terraced dose-response relations have been observed in single ligand application for enzymes, carriers, transporters, G protein-coupled receptors as well as for other receptive units. It seems that there is still a need for new models as analytical tools for such dose-responses, especially in the light of expanding di- and multi-merization of the receptive units for functionality.ResultsSelf-inhibition by drugs is analyzed in the frame-work of a theoretical homotropic two-state model, HOTSM. The model is a cubic reaction scheme based on a combination of conformational isomerization between two states within a receptive unit and ternary-complexing of two identical agonist molecules with the receptor. Concepts and terms related to self-inhibition are presented. HOTSM has seven independent parameters. Making a few simplifying assumptions narrows its analysis to initially look at four parameters. Some conclusions to be drawn are that a first level of spontaneous activity is solely determined by an isomerization constant, L. As ligand concentration rises, all seven parameters influence a second level of activity. At high ligand concentrations, a third level of activity is determined by only four of the seven constants, viz. the L constant and three intrinsic efficacy related constants, a, b, and d. The third level is given by 1/[1 + 1/(L·a·b·d)]. The third level may be above, at, or below the first and second levels. When the third level is above the first level, dose-responses may be bell-shaped, terraced, or reversed bell-shaped while when it is below the first level, dose-responses can attain forms of bell-shapes, reverse terraces, or reverse bell-shapes. To exemplify its use, the HOTSM is fitted to experimental dose-responses from sources in the literature. Development of the HOTSM is reviewed.ConclusionsThe homotropic two-state model, HOTSM, is a novel model for analyses of dose-responses at equilibrium that are co-operative or show bell-shapes of auto-antagonism.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products.

Slow sequential conformational changes in Escherichia coli ribosomes induced by lincomycin: kinetic evidence.

In a cell-free system derived from Escherichia coli, lincomycin produces biphasic logarithmic time plots for inhibition of peptide-bond formation when puromycin is used as an acceptor substrate and AcPhe-tRNA as a donor substrate. In a previous study, initial slope analysis of the logarithmic time plots revealed that the encounter complex CI between the initiator ribosomal complex (C) and lincomycin (I) undergoes a slow isomerization to C*I. During this change, the bound AcPhe-tRNA and lincomycin are rearranged to also accommodate puromycin, and this may account for the mixed noncompetitive inhibition (K(i)* = 70 microM) established at higher concentrations of the drug. The above-mentioned effect was further investigated by analyzing the late phase of the logarithmic time plots. It was found that C*I complex reacts with a second molecule of I, giving C*I(2) complex. However, the logarithmic time plots remain biphasic even at high concentrations of lincomycin, making possible the identification of another inhibition constant K(i)*', which is equal to 18 microM. The simplest explanation of this finding is to assume the existence of a second isomerization step C*I(2) <--> C*I(2'), slowly equilibrated. The determination of K(i)*' enables us to calculate the isomerization constant (K(isom) = 2.9) with the formula K(i)*' = K(i)*/(1 + K(isom)). Our results suggest that whenever a fast and reversible interaction of lincomycin with the elongating ribosomal complex C occurs, the latter undergoes a slow isomerization, which may be the result of conformational changes induced by the drug.

Onaran and Gurdal reply

Hall et al. raised two interrelated points on the article that has recently been published in TiPS1xOnaran, H.O. and Gurdal, H. Trends Pharmacol. Sci. 1999; 20: 279–286Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all References1: (1) the general behaviour of Hill coefficient in the interacting-receptor model, and (2) the discrepancy between the predictions of the model on Hill coefficient and the experimental data obtained in β2-adrenoceptor-overexpressing transgenic mice2xGurdal, H., Bond, R.A., Johnson, M.D., Friedman, E., and Onaran, H.O. Mol. Pharmacol. 1997; 52: 187–194PubMedSee all References2.We agree with their comments concerning the experimental data obtained in transgenic mice: a Hill coefficient of less than one, which cannot be predicted by the interacting-receptor model in the absence of a G-protein–receptor interaction, was indeed the case for the competition binding of isoproterenol to β2-adrenoceptor-overexpressing cardiac membranes in the presence of Gpp(NH)p. This point needs further clarification as commented on by Hall et al.However, the first point concerning the general behaviour of Hill coefficient in the model deserves some additional consideration. Unlike the case of nondissociating-multisubunit-multivalent proteins, different oligomers and the monomer in the interacting-receptor model are all in equilibrium with each other and only one of the monomeric states (active or inactive) of the receptor is assumed to enter the oligomerization equilibrium, which is reciprocally linked to the ligation equilibrium in an efficacy-dependent manner. Such a scenario brings additional complications to the expectations regarding the Hill coefficient of ligand-binding isoterms in the interacting-receptor model. Hill coefficient becomes a complicated function of receptor concentration, dimerization affinity (L), isomerization constant of monomeric receptor (J) and the efficacy parameter (β). The situation is summarized in Fig. 1Fig. 1, which shows the distribution of Hill coefficients depending on the efficacy parameters, β, at high receptor density (10−9) in the case of dimerization. Each point in the figure (10 000 points total) corresponds to random values of L, J and β, which were picked from log-uniform distributions in the following ranges: log(L)=[10,12], log(J)=[−4,2], log(β)=[−4,4]. Following points are evident in the picture: (1) Hill coefficient of neutral ligands [log(β)=0] is always 1 regardless of the initial abundance of R* (dimerization-competent monomeric state) and dimer, which are determined by J, L and the total number of receptors, (2) for active ligands [agonist or negative antagonist, log(β)≠0], the possibility that Hill coefficient >1 arises; however, (3) even for strong negative antagonists or for strong agonists [i.e. log(β)=−4 or 4, respectively], the possibility that Hill coefficient ≈1 still exists depending on the values of the other parameters. In other words, Hill slope >1 for active ligands at high receptor density is not a necessity in the model’s repertoire. Furthermore, at those points that possess Hill slopes close to 1 for active ligands (at high receptor density), it is still possible to observe affinity variation with the variation of receptor density, which implies parallel shifts in binding isotherms when receptor density is changed (not shown). Therefore, we would avoid using the Hill coefficient as a diagnostic parameter in the context of the interacting receptor model, unless the parameters J and L are known experimentally.Fig. 1Behaviour of Hill coefficient in an interacting-receptor model.View Large Image | Download PowerPoint Slide

The multiple phenotypes of allosteric receptor mutants.

Channel-linked neurotransmitter receptors are membrane-bound heterooligomers made up of distinct, although homologous, subunits. They mediate chemo-electrical signal transduction and its regulation via interconversion between multiple conformations that exhibit distinct pharmacological properties and biological activities. The large diversity of functional properties and the widely pleiotropic phenotypes, which arise from point mutations in their subunits (or from subunit substitutions), are interpreted in terms of an allosteric model that incorporates multiple discrete conformational states. The model predicts that three main categories of phenotypes may result from point mutations, altering selectively one (or more) of the following features: (i) the properties of individual binding sites (K phenotype), (ii) the biological activity of the ion channel (gamma phenotype) of individual conformations, or (iii) the isomerization constants between receptor conformations (L phenotype). Several nicotinic acetylcholine and glycine receptor mutants with complex phenotypes are quantitatively analyzed in terms of the model, and the analogies among phenotypes are discussed.

Factors Affecting Solution Properties of Vanadium(V) Compounds: X-ray Structure of β-cis-NH4[VO2(EDDA)]1

The solution properties of two isomeric forms of the V−EDDA complex (the symmetric α-cis and asymmetric β-cis) have been studied using multinuclear NMR spectroscopy. The isomerization constant of the aqueous equilibrium mixture expressed as [α-cis]/[β-cis] increased on the addition of organic solvents (methanol, formamide, and DMSO) and decreased on the addition of salts (NaCl, NaClO4, and NH4Cl). The isomerization constant and corresponding thermodynamic parameters were determined under a variety of conditions. The solution studies were consistent with the interpretation that solvation is the major contributor to isomer stability. Solid state characterization of the β-cis isomer of NH4[VO2(EDDA)] confirmed this conclusion and allowed detailed examination of the role of hydrogen bonding in this complex. The structural parameters for this complex are triclinic, P1, a = 6.787(1) A, b = 7.272(1) A, c = 11.014(1) A, α = 95.084(9)°, β = 92.805(8)°, and γ = 100.009(9)°. The dynamic processes of these complexes...

Heparin protects cathepsin G against inhibition by protein proteinase inhibitors.

Cathepsin G, a cationic serine proteinase present in neutrophils and monocytes, is able to cleave biologically important proteins and may thus participate in tissue destruction during inflammation. Its activity is physiologically controlled by the fast-acting serpins, alpha 1-anti-chymotrypsin (ka = 5 x 10(7) M-1 S-1) and alpha 1-proteinase inhibitor (ka = 2.7 x 10(5) M-1 S-1). We have shown that cathepsin G forms a tightly bound 1:1 complex with a 5-kDa heparin fragment (Kd = 1.9 x 10-8 M). The partial enzymatic activity retained by this complex is inhibited extremely slowly by the above 68- and 53-kDa serpins. The activity of the complex is also virtually resistant to inhibition by eglin c, and 8-kDa non-serpin inhibitor. A detailed kinetic investigation showed that the inhibition of heparin-bound cathepsin G by the three proteins proceeded via a two-step mechanism. [formula: see text] The three inhibitors have widely different Ki* values (0.18-13 microM) (Ki* = k-1/k1). Their isomerization constants k2 are, however, all in the same range and their extremely low values (0.7-3 ms-1) account for the very low rate of cathepsin G inhibition. The second-order inhibition rate constants k2/Ki* were 4300, 700, and 52 M-1 S-1 for alpha 1-antichymotrypsin, alpha 1-antitrypsin, and eglin c, respectively, indicating that, if heparin is present in vivo, the two former physiological inhibitors will be unable to prevent cathepsin G-mediated proteolysis. Neutrophil elastase binds the 5-kDa heparin fragment with an affinity identical to that of cathepsin G. alpha 1-Proteinase inhibitor reacts, however, much faster with heparin-elastase (ka = 1.8 x 10(6) M-1 S-1) than with heparin-cathepsin G (k2/Ki* = 700 M-1 S-1).

Factor Independent Activation of rrnB P1: An "Extended" Promoter with an Upstream Element that Dramatically Increases Promoter Strength

The extraordinary strength of the Escherichia coli rRNA promoter rrnB P1 derives primarily from sequences upstream of the core (-10, -35) region. We find that sequences between -40 and -60 increase the activity of this promoter at least 30-fold in vitro and in vivo. This region, which we refer to as the upstream (UP) element, is located between the -35 consensus hexamer and the previously characterized binding sites for the rRNA transcription factor Fis. The effect of the UP element is independent of Fis in vivo , and independent of any other proteins besides RNA polymerase (RNAP) in vitro. The UP element increases the overall second-order rate constant for association of RNAP with the promoter (ka) and probably the apparent overall first-order isomerization constant (ki). Together with the previously reported protection of the UP element region by RNAP in footprinting experiments, these results indicate that rrnB P1 has an "extended" promoter structure, consisting of the UP element and the core promoter region. We find that the UP element is a separable promoter module that can function to increase the activity of the lac core promoter in an rrnB P1-lac hybrid promoter construct. A functional UP element is not absolutely essential for stimulation of rrnB P1 by the Fis protein.

Slow-onset inhibition of ribosomal peptidyltransferase by lincomycin

In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin. N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction [1], C+ S ⇌ K S CS→ C′+ P where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C). The entire course of reaction [1] appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot). The slope of this straight line gives the apparent k S obs = k 3[ S] (K s + [ S]) . In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope ** K e= k 3[ S] {K s(1+[ I] K 1) and a late slope ** k 1= k 3[ S]{ K s(1+[ I] k′ 1+[ S]} . Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with K i = 10.0 μM. The late slopes also give competitive inhibition with a distinct inhibition constant K' i = 2.0 μM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C+ I ⇌ k 5 k 4 CI ⇌ k 7 k 6 C∗ I where the isomerization step CI ⇌ CI∗ is slower than the first step C + I ⇌ CI, K i = k 5 k 4 and K′ i = K′ i [ k 7 (k 6 + k 7) ] . Corroborative evidence for this model comes from the examination of reaction [2] alone in the absence of S. This reaction is analyzed as pseudo-first-order going toward equilibrium with eq I = k 6 + k 6 [I] (K i + [ I]) . The plot of k eq I versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k = 5.2 min −1 and k 7 = 1.3 min −1. This is the first example of slow-onset inhibition of ribosomal peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k 6 and k 7. We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA. Subsequently, as a result of a conformational change, an isomerization occurs (CI ⇌ C∗I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.