The mechanism of anion inhibition of pork heart aspartate aminotransferase

The mechanism of anion inhibition of the reaction of the pork heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1) with erythro-β-hydroxy-l-aspartate was investigated. This reaction produces a mixture of complexes, one of which is characterized by an absorption maximum at 492 nm. Spectrophotometric analysis of equilibrium mixtures of aspartate aminotransferase and erythro-β-hydroxy-l-aspartate, in different buffers, indicated that acetate, chloride, and cacodylate were competitive inhibitors of hydroxyaspartate binding. Pyrophosphate, however, was not a competitive inhibitor. Between pH 4.5 and 9.0 the affinity of the enzyme for the monovalent anions decreased as the pH increased. The data indicated that the anion binding group had a pK_a in the range from pH 6 to 7, depending upon the anion studied. From pH 4.5 to 9.0, the substrate dissociation constant and the distribution of enzyme-substrate complexes were both unaffected by pH. By stopped-flow spectrophotometry, an initial rapid relaxation ($\text{t}\text{1}\text{2}\text{= 2–8}\text{ms}$) was associated with an increase in absorbance at 492 nm, and this rate depended upon both substrate and buffer concentrations. A slower relaxation ($\text{t}\text{1}\text{2}\text{= 180}\text{ms}$) was associated with a decrease in the absorbance at 492 nm to approximately 70% of the value attained in the first rapid reaction. The rate of this slower reaction was largely independent of substrate and buffer concentrations. Kinetic analysis of the rates of the first relaxation in several different concentrations of Tris-acetate buffer of pH 8 showed that the rate of association decreased with increasing acetate concentration whereas the reaction rate for dissociation was unaffected. Thus, acetate appears to exert its inhibitory effect by preventing the formation of the enzyme-substrate complex rather than by displacing the substrate from the enzyme.     

The enhancement of cytochrome P-450 catalyzed methoxyflurane metabolism by a heat-stable microsomal protein

We report the existence of a microsomal, heat-stable, trypsin-sensitive factor that stimulates the O-demethylation of methoxyflurane (CHCl₂CF₂OCH₃) by partially purified preparations of rabbit hepatic cytochrome P-450. The factor is able to stimulate by five to twelve-fold the methoxyflurane metabolizing activity of cytochrome P-450. In contrast, the metabolism of benzphetamine is not affected by the presence of the factor. The factor is inactivated by extraction with methanol, chloroform, butanol and ethanol. It remains intact after treatment with 6M guanidine hydrochloride and is soluble in trifluoroethanol. Thus, the weight of evidence indicates that this factor is a rather hydrophobic protein.     

Electrostatic facilitation of the reaction catalyzed by the manganese-containing and the iron-containing superoxide dismutases

Both the iron-containing and the manganese-containing superoxide dismutases from Escherichia coli show diminished activity with increasing ionic strength, indicative of electrostatic facilitation of the catalyzed reaction. Since both enzymes bear a net negative charge at the assay pH, as does the substrate, this suggests a cationic locale in the active site region. Acetylation of the enzymes inverted their response to increasing ionic strength. It thus appears that lysine residues provide the observed electrostatic facilitation. A specific inhibition by large monovalent anions was observed with the iron-containing superoxide dismutase and was taken to indicate the presence of a cationic group, within a hydrophobic crevice, at the active site.     

The effects of cations and trypsin on extraction of chlorophyll-protein complexes by octyl glucoside

The extraction of chlorophyll-protein (CP) complexes from thylakoids by the detergent octyl glucoside is strongly affected by pretreatment of the thylakoids with trypsin or cations. In these experiments, washed thylakoids were incubated in the presence of 0.5 μm to 5 mm Mg²⁺, pelleted, and extracted with octyl glucoside (30 mm). Increasing amounts of Mg²⁺ depressed extractability of all CP complexes, but especially the chlorophyll a + b-containing light-harvesting complex (LHC). This cation effect is observed with other cations which promote thylakoid stacking (5 mm Mn²⁺ or Ca²⁺, 50 mm Na⁺). However, the effect is not merely due to stacking, since low concentrations of Mg²⁺ (0.5 μmto 0.5 mm) have a marked effect on extractability but have no effect on light scattering (OD 550 nm), an indicator of stacking. Furthermore, trypsin treatment of thylakoids stacked with 5 mm Mg²⁺ caused a significant reversal of stacking, but had little effect on extractability. Trypsin treatment of unstacked membranes resulted in increased extractability of all CP complexes, but especially of the LHC. Cation-treated membranes are also significantly different from those “stacked” at pH 4.5. While the latter do show decreased extractability, there is no change in the chlorophyll $\text{a}\text{b}$ ratio of the extract, and the membranes cannot be “unstacked” with trypsin. We conclude that octyl glucoside extractability reflects the lateral interaction of CP complexes with each other and with other components in the same plane of the membrane. It is clear that divalent cations have several effects on thylakoid membranes, not all of which are due to their ability to promote stacking.     

Isolation of citroylformic acid-gamma-lactone from a commerical batch of oxaloacetic acid: inhibition of apotyrosine aminotransferase conversion to holoenzyme by this substance.

Citroylformic acid-γ-lactone (CFA, 1-keto-2,4-dihydroxy-4-carboxyadipenoic acid(2–3)-1,4-lactone), isolated from a commercial batch of oxaloacetate, inhibited conversion of rat liver apotyrosine aminotransferase (EC 2.6.1.5) to holoenzyme. Using partially purified enzyme, the K_i was determined to be less than 0.7 mm. A more definitive K_i was difficult to obtain because at pH 7 CFA had a half-life of about 2 hr. Inhibition of the enzyme by CFA was stereospecific and reversible; the S (?) stereoisomer was approximately 10 times more inhibitory than its R(+) antipode, and over 90% of inhibited enzyme was recoverable after overnight dialysis. Preineubation of apotyrosine aminotransferase with its coenzyme (pyridoxal phosphate) prevented inhibition by CFA, and a substantial fraction of enzyme that had been inhibited by CFA could be readily reactivated by addition of high concentrations of pyridoxal phosphate. Studies with inhibitor analogs indicated that both a partially unsaturated lactone ring and a stereospecific carboxymethyl group are required for maximal inhibitory activity. The sodium salts of citroylformic acid and oxalopyruvic acid, formed by the hydrolysis of their respective lactones, were not inhibitory; 1-keto-2,4-dihydroxy-4-carboxyadipic acid-γ-lactone and little inhibitory activity, and 1-keto-2,4-dihydroxyglutarenoic acid-γ-lactone and 1-keto-2,4-dihydroxybutene-γ-lactone were somewhat better inhibitors than the R(+) stereoisomer of CFA. The possibility that CFA is a naturally occurring biological substance is discussed.     

Effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase

The effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase has been investigated. Citrate showed modest effect on the ATP-orthophosphate and ATP-ADP exchange reactions. In contrast, this tricarboxylic acid caused marked concentration-dependent stimulation of the acetyl CoA-malonyl CoA exchange reaction which was concomitant with the activation of acetyl CoA carboxylation. The data obtained are consistent with the suggestion that activation by citrate of the overall forward reaction (malonyl CoA synthesis) primarily reflects enhancement of the carboxyltransferase half-reaction catalyzed by rat mammary gland acetyl CoA carboxylase.     

Enzyme reaction rates and the stochastic theory of kinetics

The kinetics of an elementary reaction step are discussed from the viewpoint of the stochastic theory of chemical kinetics. The general form of the rate constant found in the stochastic approach is described, and compared with the expression from transition state theory. Whereas the stochastic theory predicts a rate enhancement in cases which are not adiabatic (in the quantum mechanical sense), transition state theory,which is essentially an adiabatic theory of reaction rates, does not permit inclusion of the effect. This effect can be expected to be of greater importance in cases of catalysis by structures, such as enzymes, containing large numbers of vibrational degrees of freedom (particularly low frequency ones) than in cases lacking such structures. The stochastic theory is more general than the transition state theory, the rate constant expression given by the latter being obtainable from the former when restrictive assumptions, including that of adiabaticity, are made. Interpretations of enzyme catalysis based on the transition state theory must thus be viewed as speculative.     

Metal chelates of heterocyclic nitrogen-containing ketones. XIV. Metal ion,anion and substituent effects on the enolization of mono-keto compounds

A number of new complexes of iron(II), cobakt(II), nickel(II), copper(II) and palladium(II) containing 2-picolyl-p-nitrophenyl- or 2-picolyl-p-tolyl ketone, L and L′, respectively, and various anions (Cl^?, Br^?, NSC^?, BF₄^? or ClO₄^?) have been synthesized and characterized by elemental analyses, magnetic susceptibility, ESR, IR and reflectance spectral measurements. The stereochemistry and the nature of the complexes are markedly dependent upon the molar of the reactants, the anions and the ligand substituents. In all complexes the ligands are cheated to the metal ion via the pryridine nitrogen and the carbonyl oxygen atoms, whereby in the case of [ML₂]X₂, M = iron(II) and [ML₃]X₂, M = cobalt(II) or nickel(II) and X = ClO₄^? or BF₄^?, the 2-picolyl-p-nitrophenyl ketone exists in its enol form which is only deprotonated in the presence of palladium(II). The ligand field parameters (Dq, B′, λ and β) are calculated and related to the electronic environment and the basicity of the ligands.     

Ultrastructural studies of the effects produced by some amino acid metal systems on escherichia coli B

Escherichia coli cells were grown in the presence of L-serine and gallium(III) nitrate a different molar ratios. Under these conditions ultrastructural changes were observed in the cells when examined under the electron microscope. Although some changes were seen inside the cell the major modifications were observed at the cell surface. These changes appeared to involve both the cell and the peptidoglycan layer. Autoradiography at the electron microscope level undertaken with similar mixtures and containing L-(3 - 3H) serine showed silver grains at or near the cell surface. In some cases, surface modifications were so pronounced that they resulted in the E. coli appearing as sheets of cells.No cell surface changes were detected when mixtures of L-serine and potassium tetrachloropalladate(II) were used as modifying agents. With the palladium(II) mixtures all changes observed were intracellular. These modifications included the appearance of membrane-bound vehicles, clumping of the cytoplasm and changes in the nucleoplasm. Autoradiography carried out in the presence of L-(3 - 3H) serine showed a significant proportion of silver grains over the nuclear region. A pure palladium(II) complex of L-serine was examined as a modifying agent in the concentration range 1–9 μ/cm³ resulting in very pronounced modification of the cells when exposed to higher concentrations.     

19F nuclear magnetic resonance observations of aldehyde dismutation catalyzed by horse liver alcohol dehydrogenase

We have examined aspects of the second catalytic activity of alcohol dehydrogenase from horse liver (LADH), which involves an apparent dismutation of an aldehyde substrate into alcohol and acid in the presence of LADH and NAD. Using the substrate p-trifluoromethylbenzaldehyde, we have observed various bound complexes by ¹⁹F NMR in an effort to further characterize the mechanism of the reaction. The mechanism appears to involve the catalytic activity of LADH · NAD · aldehyde complex which reacts to form an enzyme · NADH · acid complex. The affinity of the acid product for LADH · NADH is weak and the acid product readily desorbs from the ternary complex. The resulting LADH · NADH can then react with a second molecule of aldehyde to form NAD and the corresponding alcohol. The result is the conversion of two molecules of aldehyde to one each of acid and alcohol, with LADH and NAD acting catalytically. This sequence of reactions can also explain the slow formation of acid product observed when alcohol and NAD are incubated with the enzyme.     

Oxidation of hemoglobin by arenediazonium salts. The influence of dioxygen

The reactions of human hemoglobin with p-nitro- and p-chlorobenzenediazonium tetrafluoroborates in the presence and absence of molecular oxygen have been investigated in kinetic detail. The oxidation of iron(II) occurs with first order rate dependence on both the hemoglobin and diazonium salt concentrations, but inverse first order dependence on the concentration of molecular oxygen characterizes reactions performed in the presence of O₂. In the absence of O₂, nitrobenzene is the only product observed from hemoglobin oxidation by p-NO₂C₆H₄N₂⁺BF₄^?, and a 1:1 stoichiometry exists between nitrobenzene produced and Fe(II) oxidized. In the presence of O₂, p-nitrophenol is the dominant product, but product yield is dependent on the ratio of reactants. Electron transfer to the diazonium salt rather than its corresponding diazohydroxide or diazoate is inferred from the relative absence of pH dependence on the rate of oxidation. The composite results are consistent with a mechanism for hemoglobin oxidation that requires molecular oxygen dissociation from oxyhemoglobin prior to oxidation by the diazonium salt. Implications of this investigation for the mechanism of arylhydrazine reactions with hemoglobin are discussed.     

Thermodynamics of the reaction of ferric myoglobin from Aplysia limacina with azide and fluoride. Dependence of enthalpy changes on pH

The effect of pH on the enthalpy changes for binding of azide and fluoride to ferric myoglobin from Aplysia limacina, which lacks the distal histidine, has been investigated. Over the whole pH range explored (3.8 to 9.5), -delta H degrees values for the formation of the hemoprotein-ligand complexes are: (1) much greater than the variations in -delta G degrees; (2) always negative; and (3) show a dependence upon pH characterized by a maximum for azide and a minimum for fluoride binding, centered at pH 4.55 (identical to pHch). This value agrees well with that expected from the linear correlation between pHch and the simple function "Lys+Arg-Glu-Asp-2" proposed by Beetlestone and others. Data reported here greatly extend the pH range for which the linear correlation between the net charge of the macromolecule and pHch has been found to hold, and indicate unequivocally that the pH dependence of -delta H degree for the binding of anionic ligands does not uniquely require the presence of the histidyl residue at the distal position.     

Dinuclear complexes with a μ-cyano ligand. Part X. Synthesis and characterization of several derivatives of cis-diaquaamminecobalt(III) complexes. Influence of pH

Six new dinuclear complexes, derived from cis-[Co(H₂O)₂(NH₃)₄]³⁺, cis-[Co(H₂O)₂(en)₂]³⁺ and [M(CN)₄^2? (M = Ni, Pd, Pt) were prepared and characterized by means of chemical analysis, electronic and IR measurements. The influence of the pH on the rate of the reaction was studied for the two derivatives of [Pd(CN)₄]^2?, showing that the best conditions to obtain the dinuclear compounds are at pH near 6, where the predominant species are cis-[Co(OH)(H₂O)(amine)₂]²⁺. The [Pt(CN)₄]^2? derivatives show PtPt interactions both in the solid state and in solution.     

Steric effects on activation energy for the electrochemical oxidation of organocobaloximes

Heterogeneous electron transfer rate constants were determined as a function of electrode potential for one-electron oxidation in acetonitrile (AN) at O °C of a series of organocobaloximes [R-Co(DH)₂L] bearing widely different organic groups. Reaction entropies were determined by voltammetric half-wave potential (E^r_{$\text{1}\text{2}$}) measurements in a non-isothermal cell. The electron transfer coefficients and reorganization parameters were calculated following the Marcus theory. The reaction free energies relative to a reference couple ΔG° are linearly correlated with the polar Taft constant of the organic substituent R.The steric effects on ΔG° are shown by the correlation of E^r_{$\text{sol1}\text{2}$} with the CoC bond distance.Assuming constancy of double layer effects along the series in the given solution composition, the trends of the apparent rate constants k_app were considered in order to evaluate the effects of the nature of the organic ligand on the activation energy ΔG³ of the electron transfer. The steric effects on ΔG³ are pointed out i.a. by consideration of the relationship between ΔG³ and ΔG°.     

The copper(II) catalyzed reaction of L-ascorbinc acid with tris(oxalato)cobaltate(III) ion in aqueous solution

The kinetics of the reaction between tris(oxalato)cobaltate(III) ion and L-ascorbic acid were studied in aqueous solution over a range of copper(II) ion concentrations, ascorbic acid concentrations, pH values and temperatures. A rate law of the type rate = k[HA^?][Cu²⁺][Co³⁺], where HA^? = ascorbate, is suggested by the experimental results. A mechanism involving a copper-ascorbate complex is proposed.     

NMR coalescence effects resulting from stereochemical non-rigidity and halide exchange in octahedral rhodium(III) and iridium(III) tertiary phosphine complexes

The PMe₂Ph ligands in the aquo-cations mer- [MCl₂(H₂O)(PMe₂Ph)₃][ClO₄] rapidly exchange on the NMR time-scale giving coalescence in the ¹H and ³¹P NMR spectra. Dissociation of the H₂O ligand which is trans to PMe₂Ph leads to a five- coordinate intermediate. This intermediate (M = Rh) is believed to be involved in the rapid reaction of [RhCl₂(H₂O)(PMe₂Ph)₃] [ClO₄] with mer- [RhCl₃(PMe₂Ph)₃] by a chloride transfer mechanism leading to total exchange of the PMe₂Ph ligands.     

Model compounds for microbial iron-transport compounds. Part V. Substituent effects in the catechol/FeCl3 system

Titration data are reported for a number of substituted catechol molecules with ferric chloride. The data are interpreted in terms of the complexes formed and their stabilities. The pH's at which one complex transforms to another have been shown to have a linear relationship to modified Hammett substituent constants. It is demonstrated that this approach can be used to forecast approximate stabilities of complexes formed with iron, if the Hammett substituent constant of the ligand is known.     

The chemistry of rhenium and technetium. Part III. The synthesis and characterisation of oxo-bis-(tetrathiomolybdenato)-technetium(V) and its reduction by triphenylphosphine

The Complexes (Bu₄N)[TcO(MoS₄)₂] and Tc- (PPh₃)₂(MoS₄)₂ were prepared. The former complex has a much lower Tc-O stretching frequency than is generally found gor the TcO³⁺. moiety. The latter technetium(IV) Complex was obtained by the reduction of Tc^(v) O(MoS₄)₂^? with triphenylphosphine and also by the substitution reaction of TcCl₄(PPh₃)2 with MoS₄^2-. Previous reductions of this nature have led to the isolation of species that differ by two formal oxidation state numbers from the oxidant.     

Lysine to 13C-labeled homoarginine conversion in microbial cytochromes c. Electron transport rates and 13C nuclear magnetic resonance spectroscopy

Seven microbial and one mammalian species of cytochrome c have been reacted with O-methylisourea to convert lysine residues to homoarginines containing enriched ¹³C. This set of guanidinated cytochromes has been assayed for electron transport reactivity and the nuclear magnetic resonance spectra of incorporated label have been obtained. The set consisted of c-type cytochromes from horse, Saccharomyces cerevisiae, Candida krusei, Paracoccus denitrificans, Pseudomonas aeruginosa, Rhodospirillum rubrum, Rhodopseudomonas capsulata, and Rhodopseudomanas spheroides. All derivatives demonstrated high electron transport reactivity with cytochrome oxidases; at some concentrations this rate was 100% or higher compared to corresponding native rates. All labeled ferricytochrome spectra followed a common pattern giving about five resolved or partially resolved resonance peaks. Two of these, at approximately 158.1 and 157.3 parts per million, correspond to single carbon sites. They have been assigned to labeled lysine 27 and lysine 79 (horse numbering), respectively, on the basis of sequence comparisons and an approximate chemical shift calculation. Labeled ferrocytochrome spectra were obtained and shown to be more diverse than the set of ferric spectra. Poly-[¹³C]homoarginine was prepared and shown to be an inhibitor of the horse cytochrome c-cytochrome oxidase reaction but an activator for the reactions of Paracoccus cytochrome c550. Relaxation measurements indicated that polyhomoarginine forms a complex with both cytochromes c.