Slow Conformational Change Research Articles

Pre-steady-state kinetic analysis of the reactions of alternate substrates with dialkylglycine decarboxylase.

The pre-steady-state kinetics of the half-reactions of several substrates with dialkylglycine decarboxylase are examined by multiwavelength kinetics and global analysis. The substrates examined fall into two groups: those that exhibit simple, monophasic kinetics and those that exhibit biphasic kinetics. The rate of the AIB half-reaction is likely limited by the decarboxylation step based on the simple kinetics and spectra obtained from global analysis. The spectra for the first species in the transamination half-reactions of L-alanine and L-aminobutyrate show long-wavelength absorption characteristic of a carbanionic quinonoid intermediate. This demonstrates that formation of the external aldimine intermediates and abstraction of the C alpha protons from them are rapid. The reactions of the slower substrates L-phenylglycine and 1-aminocyclohexane-1-carboxylate may have external aldimine formation as the rate-determining step. The biphasic reactions of 2-methyl-2-aminomalonate, 1-aminocyclopentane-1-carboxylate, isopropylamine, and glycine all have external aldimine formation as the rapid observable step, based on the spectral changes observed in absorption and circular dichroism measurements. 2-Methyl-2-aminomalonate reacts approximately 10(4)-fold slower than does AIB with dialkylglycine decarboxylase, compared to approximately 10(5)-fold faster with coenzyme in solution. It is proposed that this radical reactivity reversal is due to a slow protein conformational change that is a prerequisite to decarboxylation of MAM, which occurs rapidly thereafter. Circular dichroism measurements on active site bound coenzyme provide evidence supporting this proposal. The binding of the noncovalent inhibitors pyruvate or lactate or the covalently binding inhibitor 1-aminocyclopropane-1-carboxylate all induce a slow change in coenzyme circular dichroism that quantitatively parallels the slow decarboxylation of 2-methyl-2-aminomalonate. Fast circular dichroism changes are seen in the mixing time of these measurements for both 1-aminocyclopropane-1-carboxylate and 2-methyl-2-aminomalonate, indicating rapid external aldimine formation on this longer time scale.

Effects of Adsorption Conditions on Kinetics of Protein Adsorption and Conformational Changes at Ultrafine Silica Particles

Human hemoglobin (HHb, a very soft protein) was adsorbed on ultrafine silica particles. Effects of various adsorption conditions on the kinetics of adsorption and conformational changes in HHb were studied. The conformational changes in HHb upon adsorption were monitored by both circular dichroism and absorbance spectra. The adsorption rates were measured using an aqueous two-phase system to very quickly separate the ultrafine silica particles. The rate of conformational changes upon adsorption was significantly affected by pH, ionic strength, and temperature. At high temperature, large conformational changes in HHb were accomplished soon after attachment to the surfaces. On the other hand, at low temperature and/or high ionic strength, the extent of conformational changes accompanying attachment to the surfaces was small, and slow conformational changes occurred after adsorption. Moreover, the rate of conformational changes upon adsorption decreased with increasing adsorption. Renaturation of HHb during desorption was also measured and found to be accomplished within a short time. The percentage of desorbed HHb decreased with increasing adsorption time, because the extent of conformational changes in adsorbed HHb increased. Based on these results, the general tendency of the kinetics of conformational changes in proteins upon adsorption on the solid surfaces are discussed.

Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase

The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates > 20 bp compared to a 12mer substrate, the kcat/Kmincreases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcatvalues. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-specific substrates (d ln(KAss)/d ln(cNaCl) = −2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = +1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pKaof 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.

Synthesis and Conformation of Optically Active Poly(phenyl isocyanate)s Bearing an ((S)-(α-Methylbenzyl)carbamoyl) Group

Novel optically active aromatic isocyanates, 3-((S)-(α-methylbenzyl)carbamoyl)phenyl isocyanate ((S)-3MBCPI) ([α]36525 +63°) and 4-((S)-(α-methylbenzyl)carbamoyl)phenyl isocyanate ((S)-4MBCPI) ([α]36525 +181°), were synthesized and homopolymerized or copolymerized with m-methoxyphenyl isocyanate (mMeOPI) with an anionic initiator in THF at −98 °C. Poly((S)-3MBCPI) showed a very large levorotatory specific rotation ([α]36525 −1969°) and an intense CD absorption due to a predominantly one-handed helical conformation of the polymer main chain. Copolymers of (S)-3MBCPI with mMeOPI showed much larger specific rotation than that expected from the (S)-3MBCPI content. The intensity of the specific rotation of the copolymer of [mMeOPI]/[(S)-3MBCPI] = 91/9 greatly increased with a decrease in temperature, but that of poly((S)-3MBCPI) showed almost no change, suggesting that poly((S)-3MBCPI) may take a perfectly single-handed helical conformation in solution even at room temperature. Poly((S)-4MBCPI) showed a large dextrorotatory specific rotation opposite to that of poly((S)-3MBCPI). This rotation of poly((S)-4MBCPI) gradually increased in THF with time from [α]36525 +1000° to a constant value ([α]36525 +2059°). The GPC and CD analyses of the polymer suggest that this change in specific rotation with time may be caused by a slow conformational change. Poly((S)-3MBCPI) and poly((S)-4MBCPI) exhibited chiral recognition toward (±)-1,1‘-bi-2-naphthol in 1H NMR spectroscopy.

Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with Ki and alpha values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with Ki and alpha values of 0.4 microM and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 11 microM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.

DNA Polymerase β. 5. Dissecting the Functional Roles of the Two Metal Ions with Cr(III)dTTP1

DNA polymerases catalyze the synthesis of DNA with a fidelity ranging from 1000 to 100 000.2 Although the kinetic properties of DNA polymerases have been investigated extensively,3-5 the physical origin of this high fidelity is still poorly understood. The crystal structures of DNA polymerase â (Pol â) from rat brain show the presence of two Mg2+ ions in the active site6a,b,7 (Figure 1). Functionally, it has been established that DNA polymerases require divalent metal ions for catalysis.8,9 However, no kinetic evidence that two divalent metal ions are required has been obtained for DNA polymerases, nor have the roles of the two metal ions been dissected functionally. We here report the use of Cr(III)dNTP coupled with stopped-flow fluorescence to show the requirement of two metal ions, and to dissect their functional roles in the catalysis by Pol â from rat brain (overexpressed in Escherichia coli).5 Pol â can serve as a good model for nucleotidyl transfer reactions because of its small size (39 kDa) and simplicity (without exonuclease or proof-reading activities). Using a synthetic DNA primer/template containing the 2-aminopurine nucleotide analogue (2-AP) (Figure 2A), we recently reported two phases of fluorescence changes in the stopped-flow fluorescence assay of nucleotide incorporation catalyzed by Pol â (Figure 2B).10 The fast phase was attributed to a conformational change step because its rate dependence on [dTTP] is hyperbolic. The rate of the slow phase corresponds to product formation. With a dideoxynucleotide-terminated primer, we have successfully dissected the slow fluorescence change and the chemical step, and attributed the slow phase to a conformational change that is the rate-limiting step in the reaction.10 Cr(III)‚nucleotide complexes have been extensively used as analogues of Mg(II)‚nucleotide complexes to solve enzymological problems because of their exchange-inert properties.11-13 When the Pol â‚DNA complex was mixed with â,γ-bidentate Cr(ΙΙΙ)dTTP14 in the absence of Mg2+, only the fast phase was observed (Figure 2C). However, the slow phase was restored upon addition of Mg2+ (Figure 2D). Under the latter condition the substrates were turned over, and the rate of the slow phase was comparable to the rate of product (21-mer) formation measured by the rapid chemical quench assay. In the absence of any metal ions (dTTP alone) neither fluorescence changes nor catalysis occurred. These results clearly indicate that the fast conformational change is induced by the binding of Cr(III)dTTP and the slow conformational change by the binding of Mg2+. On the basis of the crystal structure shown in Figure 1, Cr3+ and Mg2+ should occupy the metal ion sites A and B, respectively.6c The results described above, taken together with our pre-steadystate kinetic analyses reported previously, strongly support the mechanism we proposed for Pol â:10

Na+/H+ exchanger: proton modifier site regulation of activity.

The Na+/H+ exchangers (NHE1-6) are integral plasma membrane proteins that catalyze the exchange of extracellular Na+ for intracellular H+. In addition to Na+ and H+ transport sites, NHE has an intracellular allosteric H+ modifier site that increases exchange activity when occupied by H+. NHE activity is also subject to control by a variety of extrinsic factors including hormones, growth factors, cytokines, and pharmacological agents. Many of these factors, working through second messenger pathways acting directly or indirectly on NHE, regulate NHE activity by shifting the apparent affinity of the H+ modifier site to more alkaline or more acid pH. The underlying molecular mechanisms involved in the activation of NHE by the H+ modifier site are poorly understood at this time, but likely involve slow protein conformational changes within a NHE oligomer. In this paper, we present initial experiments measuring intracellular pH-dependent transition rates between active and inactive oligomeric conformations and describe how these transition rates may be important for overall regulation of NHE activity.

Effect of Calcium, Other Ions, and pH on the Reactions of Barley Peroxidase with Hydrogen Peroxide and Fluoride: CONTROL OF ACTIVITY THROUGH CONFORMATIONAL CHANGE

Transient-state kinetic analysis of compound I formation for barley grain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological function. The enzyme shows very little reaction with H2O2 at pH > 5 and exhibited saturation kinetics at higher H2O2 concentrations (kcatapp increases from 1.1 s-1 at pH 4.5 to 4.5 s-1 at pH 3.1 with an enzyme-linked pKa < 3.7 (Rasmussen, C.B., Bakovic, M., Welinder, K. G., and Dunford, H. B. (1993) FEBS Lett. 321, 102-105)). In the present paper, it is shown that the presence of Ca2+ gives rise to biphasic kinetics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typical of a classical peroxidase (K1app = 1.5 x 10(7) M-1 S-1, which is pH-independent between 3.3 and 5.0). The amount of enzyme reacting in the fast phase increases with Ca2+ concentration (Kd = 4 +/- 1 mM at pH 4.0), although it is also moderately inhibited by Cl-. The absorption spectrum of BP 1, which appears to be a five-coordinate high spin ferric in the resting state changes insignificantly in the presence of Ca2+. In the presence of Cl-, it becomes six-coordinate high spin (Kd approximately 60 mM at pH 4.0) but only if Ca2+ is also present. Fluoride binds to BP 1 with monophasic kinetics in the presence of 0-5 mM Ca2+. The activating effect of Ca2+ can be mimicked only by replacing it with Sr2+ and Ba2+ ions. Comparing these data with the crystal structure of the inactive neutral form of BP 1 (Henriksen, A., Welinder, K. G., and Gajhede, M. (1997) J. Biol. Chem. 273, 2241-2248) and similar data for wild-type and mutant peroxidases of plant and fungal origin suggests (i) a proton-induced conformational change from an inactive BP 1 at neutral pH to a low activity BP 1 form with a functional distal histidine and (ii) a Ca(2+)-induced slow conformational change (at least compared with compound I formation) of this low activity form to a high activity BP 1 with a typical peroxidase reactivity. BP 1 is the first example of a plant peroxidase whose activity can be reversibly controlled at the enzyme level by pH- and Ca(2+)-induced conformational changes.

Characterization of recombinant phytochrome from the cyanobacterium Synechocystis.

The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli. In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An approximately 300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.

Restructuring the active site of fumarase for the fumarate to malate reaction.

Changes in the active site of fumarase (yeast fumarase II) that occur when fumarate is converted to malate (E.F --> E.M) must be reversed for another cycle of reaction to take place. As shown here, recycling of the enzyme includes two proton transfers and one conformational change. These events, together with the M-off step, are variously rate-determining depending on the medium. In very low salt the release of M is limited by the conformational change. Thus, (V/Km)F decreases with increased viscosity, shown with glycerol. A variety of simple anions, such as Cl- at approximately 50 mM and F itself at low concentration, activate the dissociation of M. This nonspecific anion effect is the basis for the >4-fold apparent cooperative activation by substrate. The M-off step and the conformational change are independent and random-order events. Thus, even when M-off is made rapid the rate of recycling is inhibited by glycerol, which in 100 mM NaCl inhibits Vmax but not V/Km. The enzyme form that results when M is released is M-specific, Em. Thus mesotartarate, competitive toward M, is noncompetitive toward F. The slow conformational change required for recycling of Em is activated by Pi and chaotropic anions such as azide and thiocyanate, giving rise to a nonspecific intermediate, Emf (mesotartarate becomes competitive toward F and Britton's countertransport property disappears with these activators). Evidence is presented for the locations and rates of the two proton transfer steps required to complete the cycle.

DNA polymerase beta: multiple conformational changes in the mechanism of catalysis.

Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase beta (Pol beta), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate-limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3' end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by the binding of the catalytic Mg2+ ion. The same biphasic kinetics with different rates were also observed with the thio analog dTTPalphaS and incorrect nucleotides dATP, dGTP, and dCTP. The structural nature for the two conformational changes has been discussed in relation to the available structural information of this enzyme. The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.

Smooth muscle myosin light chain kinase, supramolecular organization, modulation of activity, and related conformational changes

It has recently been suggested that activation of smooth muscle myosin light chain kinase (MLCK) can be modulated by formation of supramolecular structures (Sobieszek, A. 1991. Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by CaM. J. Mol. Biol. 220:947-957). The present light scattering data demonstrate that the inactive (calmodulin-free) MLCK apoenzyme exists in solution as a mixture of oligomeric (2% by weight), dimeric (53%), and monomeric (45%) species at physiological ionic strength (160 mM salt). These long-living assemblies, the lifetime of which was measured by minutes, were in equilibrium with each other. The most likely form of the oligomer was a spiral-like hexamer, the dimensions of which fit very well the helical structure of self-assembled myosin filaments (Sobieszek, A. 1972. Cross-bridges on self-assembled smooth muscle myosin filaments. J. Mol. Biol. 70:741-744). After activation of the kinase by calmodulin (CaM) we could not detect any appreciable changes in the distribution of the kinase species either when the kinase was saturated with CaM or when its molar concentration exceeded that of CaM. Our fluorescent measurements suggest that the earlier observed inhibition of kinase at substoichiometric amounts of CaM (Sobieszek, A., A. Strobl, B. Ortner, and E. Babiychuk. 1993. Ca2+-calmodulin-dependent modification of smooth-muscle myosin light chain kinase leading to its co-operative activation by calmodulin. Biochem. J. 295:405-411) is associated with slow conformational change(s) of the activated (CaM-bound) kinase molecules. Such conformational rearrangements also took place with equimolar kinase to CaM; however, in this case there was no decrease in MLCK activity. The nature of these conformational changes, which are accompanied by reduction of the kinase for CaM affinity, is discussed.

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis.

Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy. Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ion-independent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH. Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metal-cation-independent chemical step, such as breaking the 5' oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.

Effect of Conformational Constraints on Gated Electron Transfer Kinetics. 2. Copper(II/I) Complexes with Phenyl-Substituted [14]aneS4 Ligands in Acetonitrile1

Kinetic studies have been conducted in acetonitrile on the electron-transfer reactions of five copper(II/I) complexes involving ligands in which either a benzene or a cyclohexane ring, or both, have been substituted into the ligand backbone of the 14-membered tetrathiamacrocycle [14]aneS(4). The specific ligands utilized in this work include 2,3-benzo-1,4,8,11-tetrathiacyclotetradecane (bz-[14]aneS(4)), 2,3-trans-cyclohexano-1,4,8,11-tetrathiacyclotetradecane (trans-cyhx-[14]aneS(4)), 2,3-benzo-9,10-trans-cyclohexano-1,4,8,11-tetrathiacyclotetradecane (bz,trans-cyhx-[14]aneS(4)), 2,3-benzo-9,10-cis-cyclohexano-1,4,8,11-tetrathiacyclotetradecane (bz,cis-cyhx-[14]aneS(4)), and 2,3-cis-9,10-trans-dicyclohexano-1,4,8,11-tetrathiacyclotetradecane (cis, trans-dicyhx-[14]aneS(4)). Each Cu(II/I)L system has been reacted with three separate reducing agents and three separate oxidizing agents to examine the effect of driving force upon the kinetic parameters. The Marcus relationship has been applied to each cross-reaction rate constant to estimate the apparent self-exchange rate constant, k(11), for each Cu(II/I)L system. For all but one of the five systems, the k(11) values obtained from the three reduction reactions are in virtual agreement with the corresponding value obtained for the oxidation reaction with the smallest driving force. As the driving force for Cu(I)L oxidation increases, a smaller k(11) value is calculated for each system. This behavior is consistent with our previously proposed dual-pathway square scheme mechanism in which a significant conformational change occurs as a separate step preceding electron transfer in the case of Cu(I)L oxidation. Although direct observation of conformationally-gated electron transfer was not attained for any of the five systems included in the current work, limits for the rate constant for conformational change have been estimated from the conditions required to change the apparent pathway for the oxidation kinetics. These limits show that the Cu(I)L complex involving a single phenyl substituent (bz-[14]aneS(4)) exhibits a much slower conformational change than do any of the other systems included in this study. The implications of this observation are discussed.

Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease.

The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.

Sodium Binding Effects on Conformational Exchange in a Diquinone Calix[4]arene

NMR studies show that a 2,4-diethoxycalix[4]arene-1,3-diquinone undergoes an unusually slow conformational change, from a predominantly anti-aryl conformation to that of a syn-aryl conformation, in the presence of Na+ ion. Dramatic changes are observed throughout the entire NMR spectral region. A novel finding is that the timescale of this conversion is unusually long, taking many hours at room temperature to complete. The kinetics of this conformational change over the range of 5−55 °C were measured in a binary solvent system. The rate constants are on the order of 10-5−10-4 s-1 in the presence or absence of Na+. These results suggest a cation effect on the equilibrium position of the anti and syn complexes rather than on the rate of interconversion. The kinetics of interconversion in the presence and absence of the salt are the same. The rate determining step corresponds to rotation of an aryl moiety through the annulus of the calixarene. Additionally, we note that the initially dominant anti-aryl conformer appears to be a kinetic product of the oxidation step of the synthesis with Tl(CF3CO2)3. Variable-temperature NMR studies in the absence of salt show a stable ∼4:1 ratio of anti to syn conformer, while the compound, as initially prepared in solution, exhibits a ratio of >50:1.

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 → Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 μs time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.

Contrasting Values of Commitment Factors Measured from Viscosity, pH, and Kinetic Isotope Effects: Evidence for Slow Conformational Changes in theD-Amino Acid Oxidase Reaction

The flavoproteinD-amino acid oxidase catalyzes the oxidation ofD-amino acids to imino acids. Previous studies of pH and isotope effets on the reaction withD-alanine as substrate have established that the enzyme substrate complex partitions forward toward carbon–hydrogen bond cleavage 10 times as fast as the amino acid dissociates and that overall turnover is limited by product release. However, theV/Kvalue forD-alanine is not affected by the solution viscosity, while theVmaxvalue is only 15% limited by diffusion. These results are interpreted as evidence for a protein conformational change between an open conformation which binds substrates and a closed complex within which catalysis occurs. Such a model is supported by the recently reported structure of the enzyme–benzoate complex (A. Mattevi, M. A. Vanoni, F. Todone, M. Rizzi, A. Teplyakov, A. Coda, M. Bolognesi, and B. Curti (1996)Proc. Natl. Acad. Sci. USA93,7496–7501).

Agonist binding to the Torpedo acetylcholine receptor. 2. Complexities revealed by association kinetics.

The binding of suberyldicholine to membrane-bound Torpedo acetylcholine receptor has been monitored by fluorescence changes of covalently bound 5-iodoacetamidosalicylic acid (IAS). At equilibrium, suberyldicholine binds to two high-affinity binding sites (Kd approximately 20 nM). Kinetic experiments reveal that there is rapid formation of an initial complex (Kd approximately 2 microM) which undergoes sequential fast (k(app) approximately 1 s(-1)) and slow (k(app) approximately 0.05 s(-1)) conformational changes. These kinetics differ from those reported for other agonists [Blanchard, S. G., Dunn, S. M. J., & Raftery, M. A. (1982) Biochemistry 24, 6258-6264] in that, for suberyldicholine, there is no evidence for a second pathway involving the binding of an additional agonist molecule. These results, considered together with the observed dissociation kinetics (accompanying manuscript), suggest that each high-affinity site for acetylcholine is made up of two subsites, which suberyldicholine is able to bridge, thus occluding the binding of a second ligand. The kinetic mechanism for acetylcholine binding has been re-examined to accommodate the complexities of the [3H]-acetylcholine dissociation kinetics and the observation that, at equilibrium, no more than two occupied binding sites are detected [accompanying manuscript: Dunn, S. M. J., & Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. It is suggested that, for each acetylcholine binding site, a second ligand is able to bind but that the ternary complex is transient since one of the two bound ligands again dissociates in the formation of the equilibrium mono-liganded complex. To further probe the physical nature of the two subsites, the binding of a series of bis-quaternary suberyldicholine analogues, (CH3)3N+CH2CH2OCO(CH2)n-COOCH2CH2N+(CH3)3, to IAS-labeled receptor preparations has been examined. Analogues in which n < 5 behave like acetylcholine, i.e., a second ligand binding pathway is observed, but longer ligands (n = 5-10) act like suberyldicholine and may be long enough to cross-link the sites.

Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency.

In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Two aminoacyl analogs [3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM] and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction. Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin. C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l. Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2. The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 [corrected]. In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when [I] approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1. Such values allow the classification of CAM analogs as slow-binding inhibitors. According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4. The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.