Assessment of the degree of disability in narcolepsy

The effect of free fatty acids (FFA) and non-enzymatic glycation on the binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA) was studied using the stopped-flow technique. The influence of FFA on the binding parameters of 25% glycated HSA depended on the type of fatty acid. The addition of stearic, oleic and linoleic acids in a concentration of 0.3 mmol/l showed no inhibitory effects on the association rate constant (k2) value for DS binding to 25% glycated HSA (k2 without FFA: 385 +/- 10 s-1, k2 with FFA > or = 385 +/- 10 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, decanoic, lauric and myristic acids) showed marked inhibitory effects for 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/- 5 s-1) and for 0.6 mmol/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate constant (k2) as well as the affinity constant (KA) of DS were markedly affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 385 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. KA decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/- 0.1 x 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10(5) M-1 for 50% glycated HSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characterization of biotinylated analogs of the calcium channel blocker omega-conotoxin

We have prepared a series of biotinylated analogs of omega-conotoxin (omega CgTx) as potent, selective markers for N-type calcium channels. At pH 9.5, reaction of omega CgTx with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24, or at both positions. Kinetic competition assays of 125I-omega CgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 x 10(6) M-1 s-1) but not dissociation (1-4 x 10(-4) s-1). Comparison with rate constants obtained for the association (1.2 x 10(7) M-1 s-1) and dissociation (5 x 10(-5) s-1) of native omega CgTx indicates that while biotinylation reduces omega CgTx potency (Kdkin = k-2/k2 = 4 pM for omega CgTx), binding of these labels to membranes is nevertheless of very high affinity (Kdkin 0.1-0.3 nM).     

In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.     

Inhibition of tolbutamide 4-methylhydroxylation by a series of non-steroidal anti-inflammatory drugs in V79-NH cells expressing human cytochrome P4502C10

1. To study the role of cytochrome P4502C10 in the metabolism of the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, phenylbutazone, fenoprofen, ibuprofen, flurbiprofen, ketoprofen and naproxen, a cell line was developed stably expressing CYP2C10 cDNA. A retroviral vector construct, containing a human CYP2C10 cDNA, was transfected in V79-NH Chinese hamster lung cells by calcium phosphate co-precipitation. Sublines stably expressing human cytochrome P450 cDNA were established by selection with the neomycin analogue G418. 2. Enzymatic activity of CYP2C10 was detected by 4-methylhydroxylation of tolbutamide. This activity was inhibited to background levels by preincubation with the CYP2C9/10 inhibitor sulphaphenazole. 3. Preincubations with the NSAIDs ketoprofen, phenylbutazone, flurbiprofen and diclofenac (all 250 microM) caused a decrease in 4-methylhydroxylation of tolbutamide (500 microM), significantly different from control values (p < 0.05). Inhibition of this activity was not seen in preincubations with the NSAIDs fenoprofen, ibuprofen and naproxen (250 microM). 4. The V79-NH CYP2C10 cell line we have developed has been shown to be a useful tool to predict drug-drug interactions.     

Active and passive factors in inferior glenohumeral stabilization: a biomechanical model

M-5011 (d-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid) is a newly developed nonsteroidal anti-inflammatory drug (NSAID) that displays potent anti-inflammatory and analgesic properties with low ulcerogenic activities in animal models. In this study, the effects of M-5011 on arachidonic acid (AA) metabolism in synovial fibroblasts from patients with rheumatoid arthritis were evaluated and compared with those of other NSAIDs in vitro. Either M-5011 or ketoprofen potently inhibited prostaglandin (PG) E2 production by cyclooxygenase (COX)-2 from exogenous AA in interleukin-1beta (IL-1beta)-stimulated cells. The IC50 values of M-5011 and ketoprofen were 4.4 x 10(-7) and 5.9 x 10(-7) M, respectively. However, diclofenac and indomethacin were one order less potent. Although the latter two drugs exhibited time-dependent and irreversible inhibition on COX-2 in IL-1beta-stimulated cells, the inhibitory effects of M-5011 and ketoprofen were reversible. PGE2 production by COX-1 from exogenous AA in non-stimulated cells was also inhibited by M-5011 with a potency less than that of ketoprofen. In addition, M-5011 inhibited [14C]AA release from prelabeled synovial cells stimulated with bradykinin. However, ketoprofen hardly affected the [14C]AA release. It is likely that the effects of M-5011 on AA metabolism are, in part, responsible for its in vivo efficacy and safety profile.     

Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors

Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).     

Interaction of benzothiadiazides with human serum albumin studied by dialysis and spectroscopic methods

The interaction of a series of benzothiadiazides with human serum albumin (HSA) was investigated by equilibrium dialysis (ED) and spectroscopic methods including circular dichroism (CD). The primary binding site of benzothiadiazides was designated site II, the diazepam site on the HSA molecule, as indicated by displacement experiments using different site-selective probes. Tyrosine and lysine amino acid residues were probably involved in the binding site of these compounds to HSA. Both electrostatic and hydrophobic interactions were found to play a role in the binding of these compounds to HSA. Among the compounds tested, chlorothiazide had the highest affinity (K1 = 5.5 x 10(4) M-1, K2 = 5.8 x 10(3) M-1). The primary binding affinity of the compounds for HSA was of the order: chlorothiazide > cyclopenthiazide > polythiazide > ethiazide > trichlormethiazide = methyclothiazde > hydrochlorothiazide. Binding was insensitive to the N-B transition of HSA. The binding site is proposed to consist of a cationic site on the surface of the HSA molecular with a hydrophobic crevice to accommodate the aromatic ring of the compounds. Positions 3 and 7 of the benzothiadiazide molecule is thought to affect the binding affinity to HSA.     

The influence of an organosulfonate on the reaction between Ellman's reagent and thiocholine

The aim of this work was to elucidate the effect of methanesulfonyl fluoride on the detection reaction for the determination of cholinesterase activity. The effect of methanesulfonyl fluoride was studied by monitoring the time course of the appearance and disappearance of the detection reaction product using a stopped-flow technique. The obtained experimental data were analyzed by progress curve analysis. It was found that the effect of methanesulfonyl fluoride results from a reaction between methanesulfonyl fluoride and the detection reaction product with the following rate constant: k1 = 5.33 x 10(-2) M-1s-1. In evaluating the effect of methanesulfonyl fluoride, the decomposition of this agent in water was included in the analysis because the decomposition considerably affected the reaction between methanesulfonyl fluoride and the product. The corresponding rate constant was found to be k2 = 4.9 x 10(-5) M-1s-1.     

Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate

MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r approximately 0.38) to dynein and can be displaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titration data. Using a multiple stepwise equilibrium model, the average values of the first two association constants are K1 = 2.7 x 10(5) M-1 and K2 = 1.8 x 10(4) M-1. This value of K1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little changed [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-affinity binding sites, K3 and K4, observed previously could not be studied with mantATP within the available protein concentrations. The alpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a moderate increase in the binding affinities. Oligomeric vanadate reduces the binding affinities by approximately 50%. Addition of TritonX-100, methanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interactions. The distinct affinities of the individual binding sites are consistent with a physiological role in regulating nucleotide binding.     

Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature

The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.     

The eosin-5-maleimide binding site on human erythrocyte band 3: investigation of membrane sidedness and location of charged residues by triplet state quenching

The triplet probe eosin-5-maleimide (EMA) is a specific inhibitor of anion transport mediated by the erythrocyte membrane protein, band 3. It was previously shown that the eosin moiety is located close to the anion binding site when EMA is covalently bound to band 3 [Pan, R.-j., and Cherry, R. J. (1995) Biochemistry 34, 4880-4888]. In the present study the electrostatic properties and membrane sidedness of the EMA binding site of band 3 were further investigated by triplet state quenching. A series of stable nitroxyl free radicals, which are characterized by different charges, and I- were used as the quenchers. Time-resolved laser spectroscopy was employed to measure the triplet lifetime of EMA. It was found that the quenching reaction between the quenchers and band 3-bound EMA follows a linear Stern-Volmer plot. The quenching rate constants (Kq) of the quenchers are in the order of NH3+-TEMPO (Kq = 6.34 x 10(6) M-1 s-1) > TEMPO-Choline+ (Kq = 2.18 x 10(6) M-1 s-1) > TEMPO (Kq = 1.13 x 10(6) M-1 s-1) > I- (Kq = 2.46 x 10(5) M-1 s-1) > pyrroline-COO- (Kq = 2.18 x 10(4) M-1 s-1). Experiments with resealed ghosts and inside-out vesicles revealed that negatively charged quenchers can only access the EMA binding site from the extracellular side of the membrane while the positively charged quenchers acted from the cytoplasmic side. The ionic strength dependence of the quenching rate constants and the effects of pH on the quenching reaction were also studied. For both TEMPO-Choline+ and I-, the Kq values decreased as the ionic strength increased, but quenching by TEMPO was independent of the ionic strength variation over the same range. It was also found that at lower pH, the I- quenching rate constant increases but the TEMPO-choline+ quenching rate constant decreases. In both cases, the dependence of quenching on pH exhibited an apparent pKa of about 6.5, which suggests the involvement of one or more histidine residues. This notion gained further support from the finding that modification of His residues of band 3 by DEPC reduced I- quenching at pH 6. On the basis of these results, it is proposed that eosin is located in the anion transport channel such that it is accessible from both sides of the membrane. Histidine residues, which have previously been proposed to lie in the anion channel, probably are located on either side of the eosin probe where they contribute to electrostatic interactions which determine the Kq values for the charged quenchers.     

Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control

Peroxidation of substrates such as ascorbic acid, pyrogallol, or ferulic acid, as well as indole acetic acid oxidation catalyzed by wheat germ peroxidase (WGP)2 C2, were found to be activated by Ca2+. This activation is independent of the stabilizing effect of structural Ca2+ reported for peroxidases. Steady state kinetics of ferulic acid oxidation catalyzed by WGP C2 showed an increase in the rate of compound I formation and of compound II decomposition in the presence of the ion, evidenced as an increase in rate constants k1, from 8.9 x 10(5) to 4.5 x 10(5) M-1 cm-1, and k3, from 4.4 x 10(5) to 1.1 x 10(6) M-1 cm-1. The dissociation constant Kd, for the cyanide derivative of the enzyme showed a marked decrease from 220 to 34 microM in the presence of Ca2+, thus implying an effect of the ion in the H2O2 binding step. In the presence of Ca2+, a conformational change in the protein was revealed by tryptophan fluorescence, providing a basis for the activation mechanism. Other peroxidases such as horseradish peroxidase and WGP C3 were not activated by Ca2+. The results suggest the existence of a physiological mechanism of control of peroxidase isozymes activity mediated by Ca2+.     

Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity

It is thought that only a limited number of residues in the extended binding pocket of coagulation proteases are critical for substrate and inhibitor specificity. A candidate residue from the crystal structures of thrombin and factor Xa (FXa) that may be critical for specificity at the S2 subsite is residue 99. Residue 99 is Tyr in FXa and Thr in activated protein C (APC). To determine the role of residue 99 in S2 specificity, a Gla-domainless mutant of protein C (GDPC) was prepared in which Thr99 was replaced with Tyr of FXa. GDPC T99Y bound Ca2+ and was activated by the thrombin-thrombomodulin complex normally. The T99Y mutant, similar to FXa, hydrolyzed the chromogenic substrates with a Gly at the P2 positions. This mutant was also inhibited by antithrombin (AT) (k2 = 4.2 +/- 0.2 x 10(1) M-1 s-1), and heparin accelerated the reaction >350-fold (k2 = 1.5 +/- 0.1 x 10(4) M-1 s-1). The T99Y mutant, however, did not activate prothrombin but inactivated factor Va approximately 2-fold better than wild type. To try to switch the specificity of FXa, both Tyr99 and Gln192 of FXa were replaced with those of APC in the Gla-domainless factor X (GDFX Y99T/Q192E). This mutant was folded correctly as it bound Ca2+ with a similar affinity as GDFX and was also activated by the Russell's viper venom at similar rate, but it cleaved the chromogenic substrates with a Gly at the P2 positions poorly. The mutant, instead, cleaved the APC-specific chromogenic substrates efficiently. The Y99T/Q192E mutant became resistant to inhibition by AT in the absence of heparin but was inhibited by AT almost normally in the presence of heparin (k2 = 3.4 +/- 0.5 x 10(5) M-1 s-1). The Y99T/Q192E mutant did not inactivate factor Va, and prothrombin activation by this mutant was impaired. These results indicate that 1) residue 99 is critical for enzyme specificity at the S2 subsite, 2) a role for heparin in acceleration of FXa inhibition by AT may involve the S2-P2 modulation, and 3) the exchange of residues 99 and 192 in FXa and APC may switch the enzyme specificity with the chromogenic substrates and inhibitors but not with the natural substrates.     

Properties of methemoglobin reductase and kinetic study of methemoglobin reduction

A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0     

Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli Rep helicase dimer

Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.     

Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5). The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).     

Critical interactions in binding antibody NC41 to influenza N9 neuraminidase: amino acid contacts on the antibody heavy chain

Antibody NC41 binds to the subtype N9 neuraminidase (NA) of influenza virus A/tern/Australia/ G70c/75 and inhibits its enzyme activity. To address the molecular mechanisms by which antibodies interact with neuraminidase and the requirements for successful escape from antibody inhibition, we made amino acid substitutions in heavy chain CDRs of NC41. Antibody proteins expressed as a single-chain Fv (scFv) fused with maltose-binding protein were assayed for binding to NA by ELISA. Association constants (Ka) for wild-type and mutant scFvs are as follows: wild type, 2 x 10(7) M-1; Asn31-->Gln, 2 x 10(7) M-1; Glu96-->Asp, 1 x 10(7) M-1; Asp97-->Lys, 6 x 10(6) M-1; and Asn98-->Gln, 8 x 10(6) M-1. The Ka for intact NC41 antibody was 4 x 10(8) M-1 in the same assay, reflecting increased stability compared to that of the scFv. Mutations in the scFv antibody had less of an effect on binding than mutations in their partners on the NA, and modeling studies suggest that interactions involving the mutant antibody side chains occur, even without taking increased flexibility into account. Asp97 forms a salt link with NA critical contact Lys434; of the four mutants, D97K shows the largest reduction in binding to NA. Mutant N98Q also shows reduced binding, most likely through the loss of interaction with NA residue Thr401. Substitution N31Q had no effect on Ka. NC41 residue Glu96 interacts with NA critical contact Ser368, yet E96D showed only a 2-fold reduction in binding to NA, apparently because the H bond can still form. Asp97 and Asn98 provide the most important interactions, but some binding is maintained when they are mutated, in contrast to their partners on the NA. The results are consistent with maturation of the immune response, when the protein epitope is fixed while variation in the antibody paratope allows increasing affinity. Influenza viruses may exploit this general mechanism since single amino acid changes in the epitope allow the virus to escape from the antibody.     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Constuction of an improved system for the determination of fidelity of polymerase in PCR

Indole-3-acetic acid (IAA) is a product of tryptophan (Trp) metabolism and is found to be markedly increased in uremic sera. IAA binding to defatted human serum albumin at 37 degrees C and pH 5, 7.4, and 8.5 was studied by equilibrium dialysis, and data were analyzed assuming two independent high affinity binding sites plus a class of low affinity sites. The estimated values of the association constant of dominant site were: 7.96 x 10(3) M-1 at pH 5, 11.57 x 10(3) M-1 at pH 7.4, and 6.30 x 10(3) M-1 at pH 8.5. The competition between IAA and Trp for albumin binding at pH 7.4 was investigated. The results suggest that one specific albumin site is common for IAA and Trp, but the data were not adequately predicted by a purely competitive scheme. A better prediction was achieved assuming that the binding of IAA to a site different from the common site inhibits Trp binding.