The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values.

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.     

Beyond the EX1 limit: probing the structure of high-energy states in protein unfolding

Hydrogen exchange kinetics in native solvent conditions have been used to explore the conformational fluctuations of an immunoglobulin domain (CD2.domain1). The global folding/unfolding kinetics of the protein are unaltered between pH 4.5 and pH 9.5, allowing us to use the pH-dependence of amide hydrogen/deuterium exchange to characterise conformational states with energies up to 7.2kcal/mol higher than the folded ground state. The study was intended to search for discreet unfolding intermediates in this region of the energy spectrum, their presence being revealed by the concerted exchange behaviour of subsets of amide groups that become accessible at a given free energy, i.e. the spectrum would contain discreet groupings. Protection factors for 58 amide groups were measured across the pH range and the hydrogen-exchange energy profile is described.More interestingly, exchange behaviour could be grouped into three categories; the first two unremarkable, the third unexpected. (1) In 33 cases, amide exchange was dominated by rapid fluctuation, i.e. the free energy difference between the ground state and the rapidly accessed open state is sufficiently low that the contribution from crossing the unfolding barrier is negligible. (2) In 18 cases exchange is dominated by the global folding transition barrier across the whole pH range measured. The relationship between hydroxyl ion concentration and observed exchange rate is hyperbolic, with the limiting rate being that for global unfolding; the so-called EX1 limit. For these, the free energy difference between the folded ground state and any rapidly-accessed open state is too great for the proton to be exchanged through such fluctuations, even at the highest pH employed in this study. (3) For the third group, comprising five cases, we observe a behaviour that has not been described. In this group, as in category 2, the rate of exchange reaches a plateau; the EX1 limit. However, as the intrinsic exchange rate (k(int)) is increased, this limit is breached and the rate begins to rise again. This unintuitive behaviour does not result from pH instability, rather it is a consequence of amide groups experiencing two processes; rapid fluctuation of structure and crossing the global barrier for unfolding. The boundary at which the EX1 limit is overcome is determined by the equilibrium distribution of the fluctuating open and closed states (K(O/C)) and the rate constant for unfolding (k(u)). This critical boundary is reached when k(int)K(O/C)=k(u). Given that, in a simple transition state formalism: k(u)=K(#)k' (where K(#) describes the equilibrium distribution between the transition and ground state and k' describes the rate of a barrierless rearrangement), it follows that if the pH is raised to a level where k(int)=k', then the entire free energy spectrum from ground state to transition state could be sampled.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.     

A statistical mechanical analysis of the effect of long-chain alcohols and high pressure upon the phase transition temperature of lipid bilayer membranes.

Long-chain n-alcohols decrease the main phase-transition temperature of lipid vesicle membranes at low concentrations but increase it at high concentrations. The nonlinear phenomenon is unrelated to the interdigitation and is analyzed by assuming that alcohols form solid solutions with solid as well as liquid phases. The biphasic response originates from the balance of the free energy difference of alcohols in the liquid and solid membranes (delta gA) and the alcohol-lipid interaction free energy difference (delta u) between the two phases. When delta gA less than 0 and delta u greater than 0, or delta gA less than delta u less than 0, the transition temperature decreases monotonously according to the increase in the alcohol concentration. When delta gA greater than 0 and delta u less than 0, or delta gA greater than delta u greater than 0, it increases monotonously. Biphasic response occurs with a minimum temperature when delta u greater than delta gA greater than 0, and with a maximum temperature when delta u less than delta gA less than 0. When the alcohol carbon-chain length becomes closer to the lipid carbon-chain length, delta u is equalized by delta gA, and the temperature minimum of the main transition is shifted to extremely low alcohol concentrations. Hence, long-chain alcohols predominantly elevate the main transition temperature and lose their anesthetic potency. High pressure decreased both delta gA and delta u. Presumably, high pressure improves the packing efficiency of liquid membranes and decreases the difference between the solid and liquid membrane properties.     

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.     

Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling.

We labeled rat adipocyte cell surface glucose transporters with an impermeable, photoreactive glucose analogue, 1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoylbenzoate (B3GL) and its radioactive tracer [3H]B3GL. The labeling did not affect glucose transporter subcellular distribution in basal and insulin-stimulated adipocytes. When basal or insulin-stimulated adipocytes were labeled with [3H]B3GL and incubated at 37 degrees C in steady state, labeled GLUT4 was rapidly reduced at the cell surface and stoichiometrically recovered in microsomes without any change in GLUT4 protein levels in either pool. The labeled GLUT4 equilibrium exchange was found to be a simple first order process describable by two first order rate constants, one for internalization (k(in)) and the other for externalization (kex). Insulin affected both rate constants, reducing k(in) by 2.8-fold and increasing kex by 3.3-fold. It is concluded that GLUT4 constantly and rapidly recycles in adipocytes between the cell surface and its storage pool, and insulin increases the cell surface GLUT4 level in rat adipocytes by modulating both the internalization and the externalization steps of constitutively recycling GLUT4.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Dynamic approach to thermotropic and inotropic phase transitions

Thermotropic and inotropic phase transitions have been analysed with a dynamic theory on a self-organization. An equation of motion of a molecular assembly with strong interactions may be approximately described as: dQ/dt' congruent to -K1Q-K3Q3, where Q is a displacement from the equilibrium point Q0(identical to 0) in a vibrational state, K1 is a transition parameter. When the parameter K1 concerned with an internal driving force (partial system) changes from positive to negative through the potential bifurcation, the system transfers to a new stable state breaking down the symmetry. Such a sign change of K1 serves as a trigger to a phase transition. Using Weiss' approximation, we have evaluated the change of K1 by a function of temperature, kappa (T-TC), and have obtained the critical temperature TC of thermotropic phase transition. We have furthermore treated inotropic phase transition caused by the binding of divalent cations like Ca2+ using the function kappa (T-beta TC), where beta is a shift parameter of the critical temperature.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.     

Thermodynamic and molecular determinants of sterol solubilities in bile salt micelles

We examined, by reverse-phase high performance liquid chromatography (HPLC), the hydrophilic-hydrophobic balance of cholesterol and 12 non-cholesterol sterols and related this property to their equilibrium micellar solubilities in sodium taurocholate and sodium glycodeoxycholate solutions. Sterols investigated exhibited structural variations in the polar function (3 alpha-OH, 3 beta-OH, 3 beta-SH), nuclear double bonds (none, delta 5, or delta 7), side chain length (C27, C28, C29) and side chain double bonds (none, delta 22, or delta 24). In general, a sterol's hydrophilic-hydrophobic balance became progressively more hydrophobic (as exemplified by increasing HPLC retention values, k') with additions of side chain methyl (C28) and ethyl (C29) groups and with 3 beta-SH substitution of the 3-OH polar function. Side chain delta 22 and especially delta 24 double bonds rendered the sterols appreciably more hydrophilic, whereas a single nuclear double bond had little influence. Sterol solubilities (24 degrees C, 0.15 M Na+) were uniformly greater in 50 mM solutions of sodium glycodeoxycholate (range 0.15 to 2.5 mM) than in equimolar solutions of the more hydrophilic bile salt, sodium taurocholate (range 0.07 to 0.67 mM). For each bile salt system, a strong inverse correlation existed between micellar solubilities of sterols and their HPLC k' values, indicating that more hydrophilic sterols had greater micellar solubilities than the more hydrophobic ones. Based upon the aqueous monomeric solubilities of cholesterol (C27) and beta-sitosterol (C29) at 24 degrees C, we derived free energy changes associated with micellar binding and found that solubilization of both sterols was more energetically favored in glycodeoxycholate solutions. Although cholesterol exhibited a higher binding affinity than beta-sitosterol in glycodeoxycholate micelles, solubilization of beta-sitosterol in taurocholate micelles was more energetically favored than cholesterol by -0.6 kcal/mol. Based upon these results we offer a thermodynamic explanation for the greater micellar solubilities of more hydrophilic sterols and suggest that the high affinity, but low capacity, of a typical phytosterol for binding to trihydroxy bile salt micelles may provide a physical-chemical basis for its inhibition of intestinal cholesterol absorption.     

An NMR investigation of electron transfer in the copper-protein, plastocyanin.

1. The proton NMR spectra of oxidised and reduced French bean plastocyanin have been recorded on a 270 MHz pulsed spctrometer. 2. The spectrum of a mixture containing the protein in the paramagnetic Cu(II) and diamagnetic Cu(I) states is a superposition of the separate spectra. When ferrirate spectra. 3. The results show that self-exchange between Cu(II)- and Cu(I)-plastocyanin is slow on the NMR time scale (kex less than 2-10(4) M-1-s-1 at 50 degrees C), and that electron transfer in the presence of ferricyanide is rapid (k greater than 1-10(5) M-1-s-1).     

Prediction of protein-protein association rates from a transition-state theory

We recently developed a theory for the rates of protein-protein association. The theory is based on the concept of a transition state, which separates the bound state, with numerous short-range interactions but restricted translational and rotational freedom, and the unbound state, with, at most, a small number of interactions but expanded configurational freedom. When not accompanied by large-scale conformational changes, protein-protein association becomes diffusion limited. The association rate is then predicted as k(a)=k(a)(0)exp(-DeltaG(el)(double dagger)/k(B)T), where DeltaG(el)(double dagger) is the electrostatic interaction free energy in the transition state, k(a)(0) is the rate in the absence of electrostatic interactions, and k(B)T is thermal energy. Here, this transition-state theory is used to predict the association rates of four protein complexes. The predictions for the wild-type complexes and 23 mutants are found to agree closely with experimental data over wide ranges of ionic strength.     

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids

A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)