Effect of substrates and coenzymes on the molecular symmetry-related properties of horse liver and yeast alcohol dehydrogenases

Thermal inactivation of horse liver alcohol dehydrogenase (LADH) exhibits the following biphasic kinetics A = Afast.e-Kfast.t + Aslow.e-Kslow.t Where A is the per cent residual activity at time t,Afast and Aslow are amplitudes (expressed as % of initial activity) and kfast and kslow first-order rate constants of the fast and slow phases, respectively. For apoenzyme, Afast = Aslow = 50% of the initial activity under all conditions of temperature and pH. On the addition of a substrate or coenzyme ligand, there is a ligand concentration-dependent increase in per cent Aslow and a decrease in kslow. At sufficiently high ligand concentration, the entire time-course of inactivation can be described as a single exponential decay. The variations of per cent Aslow and of kslow with ligand concentration are consistent with the existence of two binding sites of different ligand affinities. Inactivation of LADH by excess EDTA also exhibits a similar biphasic kinetics with Afast = Aslow = 50% of the initial activity. Addition of ethanol or NAD+ brings about a concentration-dependent decrease in kfast and kslow without affecting amplitudes of the two phases. The NAD+ concentration-dependence of this decrease is consistent with a single dissociation constant for the coenzyme. Inactivation of yeast alcohol dehydrogenase by heat or excess EDTA can be represented as a single exponential decay of activity under all conditions of temperature and pH in the absence as well as presence of ethanol or NAD+. Implications of these results for the molecular symmetry of the two oligomeric enzymes in solution are discussed.     

Biphasic recombination of photodissociated CO compound of cytochrome o(s) from Vitreoscilla

The soluble cytochrome o from Vitreoscilla contains two identical subunits and two hemes. The reduced form binds 2 mol of CO in a cooperative manner with a Hill coefficient near 2 (Tyree, B., and Webster, D. A. (1978) J. Biol. Chem. 253, 6988-6991). This carbonyl compound was photolysed with a dye laser and recombination followed at 437 or 420 nm where maximal absorbance changes were registered. Recombination kinetics were biphasic, and the fast phase was approximately 10 times the rate of the slow phase. Apparent rate constants of both phases showed a nonlinear dependence on CO concentration, respectively, in conformity with a reaction scheme which assumes the transient formation of an intermediate species in both slow and fast reactions. A study of temperature dependence of the reactions gave EA = 2.7 kcal/mol for the slow reaction and EA = 3.2 kcal/mol for the fast reaction below 23 degrees C; above this temperature the slope of the Arrhenius plot for the fast reaction became positive. Maximal rates for both phases were around pH 6.5 and fell to approximately 40% of maximal at pH 12. The binding reaction was affected by even a low concentration of sodium dodecyl sulfate (0.0025%), which changed both the kinetic constant of each phase and the relative contribution of each phase to the reaction. A model which assumes the existence of fast and slow reaction conformers in equilibrium is proposed.     

Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization

The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.     

猪心乳酸脱氢酶(H_4)在盐酸胍变性过程中失活与内源荧光变化速度的比较

本文比较了大然乳酸脱氢酶和硫酸铵稳定的乳酸脱氢酶在盐酸胍性过程式中失活与内源荧光的变化速度.酶失活表现为三相反应,即极快相,其速度常数用停流装置也无法测定;快相和慢相,1M胍变性时,此二相的一级反应速度常数分别为2.7×10~(-3)秒~(-1)和4.17×10~(-4)秒~(-1).在2M硫酸铵存在条件下,用2M胍更性时,快相和慢相的一极反应速度常数分别为6.16×10~(-3)秒~(-1)和1.88×10~(-3)秒~(-1).内源荧光强度的变化表现为二相反应,即极快相,相当酶失活的极快相,但变化幅度远小于酶失活的变化幅度;快相,相当于酶失活的快相,其速度常数为失活速度常数的1/3倍.上述结果表明,类似肌酸激酶,乳酸脱氢酶的失活速度快于酶分子整体构象的变化,相对于整个酶分子来说,活性中心的构象变化对变性剂更加敏感.     

Biphasic ATP splitting of myosin at low temperature.

Studies were carried out to elucidate the nature of biphasic ATP hydrolysis by myosin at low temperature. 1. The rate of ATP splitting decreased sharply at 3--5 min after initiation of the reaction below a critical temperature (25 degrees and 30 degrees in the presence of Ca-2+ and EDTA, respectively). On the other hand, Mg-2+-ATPase [ED 3.6.1.3] did not exhibit such biphasic kinetics. 2. The Arrhenius plot of the second phase of the reaction after the rate transition gave a straight line whether the temperature of assay was above or below the critical one, giving 5.7 kcal/mole as the activation energy of Da-2+-ATPase showed features similar to those of Ca-2+-ATPase. 3. Michaelis constants for the two phases at 8 degrees were also different. In addition, the first phase of EDTA-ATPase was shown to have two different constants, depending on ATP concentration. 4. The profiles of the dependence of ATPase activity on KCl concentration were essentially the same for both phases, while bending of the time curve was scarecly observed obove pH 8 for Ca-2+-ATPase or at pH 6 for EDTA-ATPase. 5. 2, 4-Dinitrophenol abolished the phase transition for Ca-2+-ATPase and EDTA-ATPase, and heat treatment also minimized the transition for the former.     

Ferricytochrome c. Refolding and the methionine 80-sulfur-iron linkage

The refolding of urea-denatured horse heart ferricytochrome c in the presence of imidazole, 0.5 M, pH 7.0, has been examined using stopped-flow and equilibrium measurements at 407.5 nm. Thermodynamically, imidazole-cytochrome c folds and unfolds via a single transition with [urea]1/2 of 5.9 M. Kinetically, the refolding is a triphasic process: (i) a slow, urea-independent phase, time constant of 22 +/- 6 s, and an amplitude of 10-13%; (ii) an intermediate reaction, with a slightly positive urea-dependent rate constant, average time constant of 150 ms; and (iii) a fast phase with negative urea dependence of the rate constant from 4-6 M urea and positive dependence above the 6 M concentration, with the largest time constant, 25 +/- 6 ms, at 5.8 M urea, the midpoint of the transition. The amplitudes of the intermediate and the fast phases exhibit inverse dependence on the final urea concentrations, favoring the intermediate form at higher concentrations, while maintaining an almost constant sum of the two amplitudes throughout the range. The temperature dependence of the three apparent rate constants for the refolding from denatured base-line to midpoint of the transition, 9 to 6.03 M urea, yields linear Arrhenius plots with activation energies of 14, 19, and 23 +/- 3 kcal/mol for the slow, intermediate, and rapid reactions, respectively. These findings show that the slow reaction, time constant in decaseconds , does not require, directly or indirectly, the coordination of Met-80-S to heme iron. The formation of this linkage during the folding of the urea-denatured protein in the absence of extrinsic ligand, however, does alter the course of the refolding process. From a comparison of the proposed mechanisms and of the kinetic parameters for the folding of urea-denatured and of guanidine hydrochloride-denatured ferricytochrome c, it has been suggested that the two systems are distinct in detail, although both systems exhibit the slow, decasecond process.     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.     

A quantitative treatment of the kinetics of the folding transition of ribonuclease A.

New experimental data and a quantitative theoretical treatment are given for the kinetics of the thermal folding transition of ribonuclease A at pH 3.0. A three-species mechanism is used as a starting point for the analysis: U1 (slow) in equilibrium U2(fast) in equilibrium N, where U1 and U2 are two forms of the unfolded enzyme with markedly different rates of refolding and N is the native enzyme. This mechanism is based on certain facts established in previous studies of refolding. The kinetics of unfolding and refolding show two phases a fast phase and a slow phase, over a range of temperatures extending above the transition midpoint, Tm. The three-species mechanism can be used in this range. At higher temperatures a new much faster kinetic phase is also observed corresponding to the transient formation of a new intermediate (I). Although the general solution for a four-species mechanism is complex it is not difficult to extend the three-species analysis for the special case found here, in which the fast reaction (I in equilibrium N) is well separated from the other two reactions. At temperatures below the transition zone the slow phase of refolding becomes kinetically complex. No attempt has been made to extend the analysis to include this effect. The basic test of the three-state analysis is the prediction as a function of temperature of alpha2, the relative amplitude of the fast phase, both for unfolding and refolding. At temperatures above Tm for which the three-state analysis must be extended to include the new intermediate I, a crresponding quanitity alpha2(cor) is predicted and compared with measured values. Data used in the three-state prediction are values of tau2 and tau1, the time constants of the fast and slow kinetic phases, plus a single value of alpha2 measured when tau2 and tau1 are well separated. The observed and predicted values of alpha2 agree within experimental error. The analysis predicts correctly that, for these experiments, alpha2 should have the same value in unfolding as in refolding in the final conditions. The analysis also predicts satisfactorily the equilibrium transition curve from kinetic data alone. Four striking properties of the kinetics are explained or correlated by the analysis: (a) the drop in alpha2 to a minimum near Tm as well as the delayed rise in alpha2 above Tm;(b) the vanishing of alpha1 above the transition zone; (c) the sharp drop in tau1 inside the transition zone followed by a partial leveling off outside this zone; and (d) the passage of tau2 through a maximum near Tm. Through a comparison of observed and predicted values of alpha2, the analysis also rules out the alternative three-species mechanism U1 (slow) in equilibrium N (fast) in equilibrium U2. Finally, the temperature dependence of the amplitude for the fast reaction (I in equilibrium N) is discussed; the behavior of I is like that of U2 and I may be an unfolded species populated at equilibrium...     

Simulation of axoplasmic transport

We have analysed a kinetic model of axonal transport by simulating experimental tracer profiles. The existence of three phases of axoplasmic transport is assumed: fast anterograde, slow anterograde and retrograde. Each phase has its characteristic velocity. Transported materials are postulated to shift between these phases. Also catabolism and sequestration of material is allowed for in our model. Thus, we have set up equations which contain axonal transport, diffusion and cross-over terms. The rate constants of material shifts were determined by computer fitting to experimental data. Best-fitted values of the rate constants for transfer of material between the fast and slow phases were both 2 X 10(-5) sec-1, while the rate constants for transfer between the fast and retrograde phases were both 1 X 10(-5) sec-1. The rate constant of material loss from the slow phase to the extracellular space was 1 X 10(-6) sec-1. The material shift between the slow and retrograde phases was negligibly small. These data show that there is exchange of material between the fast and slow phases and between the fast and retrograde phases. However, there is no significant exchange between the slow and retrograde phases. Diffusion was found to have only a minor effect on the profiles. The velocity of the fast anterograde track in cold-blooded animals was predicted to be around 200 mm/day, or, in other words, to be close to experimentally observed values of the fast anterograde component of axonal transport.     

pH-dependent conformational transformation in mung bean glyceraldehyde-3-phosphate dehydrogenase

In thermal inactivation at pH 7.3 and below, the tetrameric apo-glyceraldehyde-3-phosphate dehydrogenase of mung beans lost half of its activity more rapidly than the rest, suggesting a pairwise arrangement of subunits (or a C2 symmetry). At pH 8.6, the activity was lost in a single exponential decay, characteristic of functional identity of sites as in a tetrahedral arrangement of subunits (or a D2-type symmetry). At intermittent pH values, the kinetics of thermal inactivation were consistent with the presence of a mixture of C2- and D2-symmetry conformations. In "sudden pH change" experiments, the observed thermal inactivation kinetics were characteristic of the final pH at which the enzyme was heated. Thus, the interconversion of the two conformations is facile and very fast. There is no gross change in molecular weight of the enzyme between pH 7.3 and 8.6.     

Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence

The kinetics of Ca2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s-1 and 200 s-1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s-1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10(9) M-1 s-1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca2+ indicators, fluo-3 is well suited for investigations of Ca2+ oscillations in biological systems.     

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

Structure-function relationship in Escherichia coli initiation factors. Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy

The reactions of rabbit muscle pyruvate kinase with 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSBA) and 5′-p-fluorosulfonylbenzoyl guanosine (5′-FSBG) from pH 7.0 to 8.0 exhibit biphasic inactivation kinetics. These reactions are characterized by three events: a fast reaction yielding partially active enzyme (with 67% of its original activity for the 5′-FSBA reaction and 45% for the 5′-FSBG reaction) which is reactivated by dithiothreitol, and two slower reactions yielding fully inactive enzymes; the product of only one of the two slower reactions is reactivated by dithiothreitol. These reactions are termed fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive inactivations. The rates of all three phases of the reactions with 5′-FSBA and 5′-FSBG increase as the pH is raised. The 5′-FSBG reaction can be described in terms of initial reaction with a single ionizable group of pK_a 7.80, 8.60, and 7.94 for the fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive reactions, respectively; pH-independent rate constants of 0.173, 0.133, and 0.0165 min^?1 are calculated for these three phases of the overall reaction. The pH dependence of the dithiothreitol-insensitive inactivation by 5′-FSBA coincides with that for 5′-FSBG, but the data for the dithiothreitol-sensitive reactions with 5′-FSBA indicate that the reaction in each phase occurs at more than one site over the pH range tested. Differential protection by ligands against inactivation by 5′-FSBA and 5′-FSBG at pH 7.4 and 8.0 indicates that, for the fast dithiothreitol-sensitive reactions, the cysteine residues participating in the two reactions are not identical, although in both cases modification has been attributed to formation of a disulfide. For 5′-FSBA, the partial inactivation appears to result from modification of cysteine residues at the noncatalytic nucleotide site, whereas for 5′-FSBG the inactivation is due to modification within the catalytic metal-nucleotide site. Reaction with 5′-FSBG seems to occur at the same locus for both the fast and slow dithiothreitol-sensitive phases, with the rate difference being ascribable to negative cooperativity among subunits. For the slow dithiothreitol-sensitive inactivation by 5′-FSBA, protection by Mg²⁺ and by Mg²⁺ plus ADP suggests that the targets of modification include the active-site cysteine that is modified by 5′-FSBG. The dithiothreitol-insensitive inactivation, shown to be due to reaction of 5′-FSBA with a tyrosine, may result from reaction of both nucleotide analogs with the same residue, although differential protection by the natural ligands suggests that 5′-FSBA and 5′-FSBG bind to two subsites within the active site.     

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296–303, 1998. © 1998 Wiley-Liss, Inc.     

Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase

Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methotrexate concentration indicates that the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 X 10(7) M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 X 10(5) M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic inhibition of acid-induced protein denaturation by trehalose and NaCl: Thermodynamic and kinetic studies

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.     

Molecular Asymmetry in Urease of Water Melon (Citrullus vulgaris)

Kinetics of urease-catalysed urea hydrolysis follows Arrhenius equation in the temperature range 10-50°C and shows an energy of activation equal to 7.14 kcal/mol. The kinetics of thermal inactivation of the enzyme is biphasic, In that half of the initial activity is destroyed more rapidly than the remaining half. The data are consistent with the rate equation: A_t = A_fast·e^-k _fast ^-t + A_slow ·e^-K _slow ^-t where A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. A similar activity decay (namely blphaslc) is also observed on storing the enzyme at +4 and ?4OC. The data suggest the existence of half-and-half distribution of sites which is a manifestation of a pre-exlstent site heterogeneity in the oligomeric protein molecule.