Complex formation between reduced D-amino acid oxidase and pyridine carboxylates

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.     

Proton release during the reductive half-reaction of D-amino acid oxidase

Changes in the net protonation of D-amino acid oxidase during binding of competitive inhibitors and during reduction by amino acids have been monitored using phenol red as a pH indicator. At pH 8.0, no uptake or release of protons from solution occurs upon binding the inhibitors benzoate, anthranilate, picolinate, or L-leucine. The Kd values for both picolinate and anthranilate were determined from pH 5.4 to 9.0. The results are consistent with a single group on the enzyme having a pK of 6.3 which must be unprotonated for tight binding, as is the case with benzoate binding (Quay, S., and Massey, V. (1977) Biochemistry 16, 3348-3354) and with tight binding of the inhibitor form with an unprotonated amino group. Upon reduction of the enzyme by amino acid substrates, two protons are released to solution. The first is released concomitantly with reduction to the reduced enzyme-imino acid charge transfer complex. The second is released only upon dissociation of the charge transfer complex to free reduced enzyme and imino acid. The first proton is assigned as arising from the amino acid group and the second from the amino acid alpha-hydrogen. These results are consistent with the flavin in reduced D-amino acid oxidase being anionic.     

Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme. It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH. However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme. The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction. We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction.     

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.     

Comparison of acute absorption of commercially available chromium supplements

Chromium (Cr) supplements are available as picolinate, nicotinate or chloride (the latter primarily in multivitamin-mineral supplements). The picolinate form has been reported to be the best absorbed and most efficacious, but some reports question which form has superior absorption. The present study examined acute Cr absorption, based on 24h urinary Cr values, for picolinate, two types of nicotinate, and chloride in young adult, non-overweight females. College-aged women were given 200 microg of Cr as each of the four supplement types in random order accompanied by a small standardized meal, separated by at least a week washout. Cr picolinate produced significantly higher 24h urinary Cr than either of two nicotinate supplements or Cr chloride given in a multivitamin-mineral supplement. This difference was seen for absolute values of the urinary Cr and for percent increases. In conclusion, based on an indirect measure of acute absorption, Cr picolinate was superior to three other Cr complexes commonly sold as supplements.     

Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells.

The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.     

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.     

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.     

Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.     

Unusual secondary specificity of prolyl oligopeptidase and the different reactivities of its two forms toward charged substrates.

Prolyl oligopeptidase belongs to a new family of serine proteases which contains both exo- and endopeptidases, and this suggests that the enzyme binds its substrate in a special manner. Its secondary specificity, i.e., its interaction with the other residues linked to the proline that accounts for the primary specificity, has been investigated by using peptide substrates of various length and charge. Elongation of the classic dipeptide substrate Z-Gly-Pro-2-naphthylamide with 1-3 residues (Gln, Ala-Gln, Ala-Ala-Gln, and Ala-Lys-Gln) resulted in decreased specificity rate constants. This indicated a limited binding site for prolyl oligopeptidase, a major difference from the finding with other serine endopeptidases. Insertion of charged residues into the substrates, such as lysine or aspartic acid, considerably affected the rates and the pH-rate profiles. The rate constants were higher with the positively charged peptides and lower with the substrates bearing a negative charge. These electrostatic effects were reduced at high ionic strength. The results can be interpreted in terms of a negatively charged active site, which exists at high pH and exerts electrostatic attraction or repulsion toward charged substrates. The pH dependencies of the rate constants with neutral substrates exhibited roughly bell-shaped curves, whereas with charged substrates the existence of two active enzyme forms was clearly demonstrated. The physiologically competent high pH form preferred positively charged substrates (Z-Lys-Pro-2-(4-methoxy)naphthylamide, Z-Ala-Lys-Gln-Gly-Pro-2-naphthylamide), whereas the low pH form reacted faster with the negatively charged substrate (Z-Asp-Gly-Pro-2-naphthylamide).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite.

Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.     

The interaction of liver alcohol dehydrogenase with phenyl adenine dinucleotide, a novel analog of pyridine nucleotide coenzymes.

We have synthesized phenyl adenine dinucleotide (P1-adenosine-5')-P2-(beta-D-ribofuranosylbenzene-5')-pyrophosphate (PhAD), a novel analog of pyridine nucleotide coenzymes. This compound contains a planar aromatic ring, as does NAD+, but lacks a positive charge. PhAD is an inhibitor of horse liver alcohol dehydrogenase, competitive with NADH. PhAD is very similar to NAD+ sterically since both compounds have a planar aromatic ring. However, PhAD resembles NADH in binding to the enzyme because the dissociation constants of both compounds show a parallel increase as the pH is raised, whereas those of NAD+ behave in the opposite manner. These observations indicate that the enzyme differentiates between NAD+ and NADH on the basis of the positive charge on the molecule and not the stereochemical orientation of the reduced nicotinamide ring.     

Kinetics of local anesthetic inhibition of neuronal sodium currents. pH and hydrophobicity dependence.

This study assesses the importance of local anesthetic charge and hydrophobicity in determining the rates of binding to and dissociation from neuronal Na channels. Five amide-linked local anesthetics, paired either by similar pKa or hydrophobicity, were chosen for study: lidocaine, two tertiary amine lidocaine homologs, a neutral lidocaine homolog, and bupivacaine. Voltage-clamped nodes of Ranvier from the sciatic nerve of Bufo marinus were exposed to anesthetic externally, and use-dependent ("phasic") block of Na current was observed. Kinetic analysis of binding (blocking) rates was performed using a three parameter, piecewise-exponential binding model. Changes in extracellular pH (pHo) were used to assess the role of drug protonation in determining the rate of onset of, and recovery from, phasic block. For those drugs with pKa's in the range of pHo tested (6.2-10.4), the forward binding rate during a depolarizing pulse increased at higher pH, consistent with an increase in either intracellular or intramembrane concentration of drug. The rate for unbinding during depolarization was independent of pHo. The dissociation rate between pulses also increased at higher pHo. The pHo dependence of the dissociation rate was not consistent with a model in which the cation is trapped relentlessly within a closed channel. Quantitative estimates of dissociation rates show that the cationic form of lidocaine dissociates at a rate of 0.1 s-1 (at 13 degrees C); for neutral lidocaine, the dissociation rate is 7.0 s-1. Furthermore, the apparent pKa of bound local anesthetic was found to be close to the pKa in aqueous solution, but different than the pKa for "free" local anesthetic accessible to the depolarized channel.     

Chromium picolinate increases membrane fluidity and rate of insulin internalization.

The effects of chromium chloride, chromium nicotinate, and chromium picolinate on insulin internalization in cultured rat skeletal muscle cells was examined. Insulin internalization was markedly increased in cells cultured in a medium that contained chromium picolinate and the increased internalization rate was accompanied by a marked increase in the uptake of both glucose and leucine. The effect was specific for chromium picolinate since neither zinc picolinate nor any of the other forms of chromium tested was effective. The increased insulin internalization rate may result from an increase in membrane fluidity since chromium picolinate and to a lesser extent, chromium nicotinate, increased the membrane fluidity of synthetic liposomal membranes.     

Conformational aspects of inhibitor design: enzyme-substrate interactions in the transition state.

The theory of absolute reaction rates suggests that enzymes, like other catalysts, can enhance the rate of a reaction only to the extent that they bind the altered substrate in the transition state (S++) more tightly than they bind the substrate in the ground state (S). ES dissociation constants commonly fall in the physiological range, but recent kinetic studies indicate that formal ES++ dissociation constants of less than 10(-20) M are achieved by enzymes of several classes. Studies with stable analogues suggest that these remarkable powers of discrimination involve a tendency of the enzyme to close around S++ in such a way as to maximize binding contacts; that several parts of the substrate contribute to S++ binding; and that their contributions to binding affinity can be strongly synergistic.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

The use of auramine O to study ligand binding and subunit cooperativity of lactate dehydrogenase

The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit.