Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c'

The kinetics of the reversible binding of cyanide by the ferric cytochrome c' from Chromatium vinosum have been studied over the pH range 6.9-9.6. The reaction is extremely slow at neutral pH compared to the reactions of other high-spin ferric heme proteins with cyanide. The observed bimolecular rate constant at pH 7.0 is 2.25 X 10(-3) M-1 s-1, which is approximately 10(7)-fold slower than that for peroxidases, approximately 10(5)-fold slower than those for hemoglobin and myoglobin, and approximately 10(2)-fold to approximately 10(3)-fold slower than that recently reported for the Glycera dibranchiata hemoglobin, which has anomalously slow cyanide rate constants of 4.91 X 10(-1), 3.02 X 10(-1), and 1.82 M-1 s-1 for components II, III, and IV, respectively [Mintorovitch, J., & Satterlee, J. D. (1988) Biochemistry 27, 8045-8050; Mintorovitch, J., Van Pelt, D., & Satterlee, J. D. (1989) Biochemistry 28, 6099-6104]. The unusual ligand binding property of this cytochrome c' is proposed to be associated with a severely hindered heme coordination site. Cyanide binding is also characterized by a nonlinear cyanide concentration dependence of the observed rate constant at higher pH values, which is interpreted as involving a change in the rate-determining step associated with the formation of an intermediate complex between the cytochrome c' and cyanide prior to coordination. The pH dependence of both the binding constant for the formation of the intermediate complex and the association rate constant for the subsequent coordination to the heme can be attributed to the ionization of HCN, where cyanide ion binding is the predominant process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceleration of the NAD cyanide adduct reaction by lactate dehydrogenase: the equilibrium binding effect as a measure of the activation of bound NAD

The binary complex of NAD and lactate dehydrogenase reacts reversibly with cyanide to produce a complex (E X NAD-CN) whose noncovalent interactions are similar to those in the E X NADH complex (where E is one-fourth of the tetrameric dehydrogenase). The reaction apparently is a simple bimolecular nucleophilic addition at the 4 position of the bound nicotinamide ring; viz., cyanide does not bind to the enzyme prior to reaction. The value of the dissociation constant for E X NAD-CN is about 1 X 10(-6) M and is independent of pH over the range of 6-8. The equilibrium constant for the reaction of cyanide with E X NAD is about 400-fold larger than that for the nonenzymic process after a statistical correction. This increment in Ke is accounted for by a 220-fold increase in the rate of the forward enzymic reaction (20 M-1 s-1) as compared with an approximately 2-fold decrease for the reverse process (9 X 10(-5) s-1). Thus, the increased value of the rate constant for bond formation in the enzymic reaction is attributed to an equilibrium binding effect that is translated almost entirely into a rate effect on that step (bond formation). Since the nonenzymic reaction is sensitive to solvent composition, this equilibrium binding effect likely is produced by environmental effects at the nicotinamide/dehydronicotinamide part of the coenzyme binding site on the enzyme.     

Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine).

Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide.

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.     

Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium

The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin-actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1.     

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.     

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

pH-dependent rate of formation of the gelsolin-actin complex from gelsolin and monomeric actin

The assembly of gelsolin with actin was followed by the increase of the fluorescence intensity of a fluorescence label bound to actin. The time course of the formation of the gelsolin-actin complex in the presence of micromolar [Ca2+] could be quantitatively interpreted by a model in which one actin molecule binds slowly to gelsolin in a rate-determining step and subsequently a second actin molecule is bound at least 40 times more rapidly. The rate of binding of the first actin molecule to gelsolin was found to be remarkably slow and to depend on the pH. The rate constants of formation of the gelsolin-actin complex range from 1.5 X 10(4) M-1 s-1 at pH 8 to 7 X 10(4) M-1 s-1 at pH 6.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

Laser flash photolysis studies of electron transfer between ferredoxin-NADP+ reductase and several high-potential redox proteins

Complex formation and the kinetics of electron transfer between ferredoxin-NADP+ reductase (FNR) and two structurally homologous acidic 4Fe-4S high-potential ferredoxins (HiPIP's) from Ectothiorhodospira halophila (HP1 and HP2) and two structurally homologous cytochromes c2 from Paracoccus denitrificans and Rhodospirillum rubrum (PC2, and RC2, respectively) have been investigated by gel filtration and laser flash photolysis techniques. Gel filtration studies indicated that complex formation occurred between FNRox and HP1ox or HP2ox at low ionic strength (10 mM) and that the complexes were completely dissociated at high ionic strength (310 mM). Laser flash photolysis using lumiflavin as the reductant demonstrated that both free HP1ox and HP2ox reacted primarily with the anionic form of fully reduced lumiflavin (LFH-), whereas FNR was unreactive. Second-order rate constants of 1 X 10(6) and 0.8 X 10(6) M-1 s-1 were obtained for these reactions at 10 mM ionic strength. Increasing the ionic strength to 310 mM resulted in an approximately 1.5-fold increase in the rate constant. Inclusion of stoichiometric amounts of FNRox into the reaction mixture at low ionic strength led to a 2.5-fold increase in the rate constants. The reaction of 5-deazariboflavin semiquinone (5-dRf.) with the oxidized HiPIP's was also investigated by laser flash photolysis. Second-order rate constants of 3.0 X 10(8) M-1 s-1 (HP1) and 2.5 X 10(8) M-1 s-1 (HP2) were obtained for the free proteins at 10 mM ionic strength. Under the same conditions, 5-dRf. reacted with free FNRox, resulting in the formation of the neutral protein-bound semiquinone (FNR.), with a second-order rate constant of 6 X 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state kinetics of the catalase reaction in the presence of cyanide

Under carefully controlled experimental conditions, the Michaelis constant for H2O2 was measured to be 1.39 and 1.29 M in the reactions of beef erythrocyte and liver catalases, respectively. These values remained unchanged at temperatures between 1 and 26 degrees C. The turnover number of the Michaelis complex was about 2.25 X 10(7) s-1 for either enzyme at 26 degrees C. The cyanide inhibition in the catalase reaction has been reported to be noncompetitive in spite of the fact that cyanide and H2O2 compete for the same site on the catalase molecule. At high concentrations of H2O2, however, the inhibition became clearly competitive. The existence of the Michaelis complex and the anomalous features of cyanide inhibition were clearly accounted for on the basis of simple kinetic models. At H2O2 concentrations below 100 mM, the catalase reaction obeyed first order kinetics with respect to H2O2 and its apparent second order rate constant was measured to be 7.6 X 10(6) and 7.9 X 10(6) M-1 . S-1 for erythrocyte and liver catalases, respectively.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study

The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation/inactivation and uni-site catalysis by the reconstituted ATP-synthase from chloroplasts

The proton-translocating ATP-synthase of chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. CF0F1 can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced states is 7.10(2) M-1.s-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10(5) M-1.s-1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7.10(-3) M-1.s-1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF1 (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100). The rate constant for ATP binding is 10(6) M-1.s-1, the 'equilibrium constant' on the enzyme EADPPi/EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s-1 and o.1 s-1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF1.     

Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase

N-Hydroxy-N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductoisomerase. In the presence of Mg2+ or Mn2+, IpOHA inhibits the enzyme in a time-dependent manner, forming a nearly irreversible complex. Nucleotide, which is essential for catalysis, greatly enhances the binding of IpOHA by the reductoisomerase, with NADPH (normally present during the enzyme's rearrangement step, i.e., conversion of a beta-keto acid into an alpha-keto acid, in either the forward or reverse physiological reactions) being more effective than NADP. In the presence of Mg2+ and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism, with an initial inhibition constant of 160 nM and a maximum rate of formation of the tight, slowly reversible complex of 0.57 min-1 (values that give an association rate of IpOHA, at low concentration, of 5.9 X 10(4) M-1 s-1). The rate of exchange of [14C]IpOHA from an enzyme-[14C]IpOHA-Mg2(+)-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days (150 h). This dissociation rate (1.3 X 10(-6) s-1) and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM. By contrast, in the presence of Mn2+ and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 X 10(4) M-1 s-1 and 3.2 X 10(-6) s-1 (half-time = 2.5 days), respectively, which define an overall dissociation constant of 38 pM. In the presence of NADP or in the absence of nucleotide (both in the presence of Mg2+), the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively. In the absence of Mg2+ or Mn2+, IpOHA does not exhibit time-dependent inhibition of the reductoisomerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)