Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: invertase-catalyzed hydrolysis of sucrose

Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived. Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 degrees C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state. Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system [J. M. Nelson and M. P. Schubert (1928) J. Amer. Chem. Soc. 50, 2188-2193] also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies.     

Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase

During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by alpha-chymotrypsin: a model system with preexistence of the isomerization equilibrium

The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine alpha-chymotrypsin have been investigated. In studies at pH 7 and 20 degrees C the presence of 0.5 M sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 degrees C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 mM) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.     

X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity

The 2.4 A resolution X-ray crystal structure of D100E trichodiene synthase and the 2.6 A resolution structure of its complex with inorganic pyrophosphate are reported. The D100E amino acid substitution in the so-called "aspartate-rich" motif does not result in large changes to the overall structure of the enzyme. In the pyrophosphate complex, however, pyrophosphate coordinates two Mg(2+) ions at the mouth of the active site without causing large changes in the structure of the enzyme. This contrasts with pyrophosphate binding in the wild-type enzyme, where pyrophosphate coordinates three Mg(2+) ions and triggers a significant conformational change that closes the mouth of the active site and optimizes packing density in the enzyme-substrate complex. The attenuation of active site closure in D100E trichodiene synthase compromises enzyme-substrate packing density and confers additional spatial and conformational degrees of freedom on the substrate and carbocation intermediates, which in turn results in the formation of five alternate sesquiterpene products in addition to trichodiene. By extension, then, the diversity of terpene cyclases in biology may have evolved in part by amino acid substitutions that fine-tune structural changes dependent on metal-diphosphate complexation that govern the formation of the active site template and enzyme-substrate packing density.     

Urea-induced denaturation of human phenylalanine hydroxylase

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of the fusion partner, the effects of increasing urea concentrations on enzyme activity, aggregation, unfolding, and refolding were examined. At pH 7.50, purified human phenylalanine hydroxylase is transiently activated in the presence of 0-4 M urea but slowly inactivated at higher denaturant concentrations. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phenylalanine (L-Phe) shifts the transition midpoint of the first transition from 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant differences in dDeltaG(D)/d[urea] (m(D) values) were observed for the first denaturation transition. In the absence of substrate, a high rate of non-covalent aggregation was observed for the enzyme in the presence of 1-4 M urea. All three tryptophan residues in the enzyme (Trp-120, Trp-187, and Trp-326) were mutated to phenylalanine, either as single mutations or in combination, in order to identify the residues involved in the spectroscopic transitions. A gradual dissociation of the native tetrameric enzyme to increasingly denatured dimeric and monomeric forms was demonstrated by size exclusion chromatography in the presence of denaturants.     

Effects of high pressure on the single-turnover kinetics of the carbamylation of cholinesterase

Pressure, as a perturbing variable, is one of the most powerful tools to investigate the thermodynamic parameters of chemical reactions and to study the mechanism of enzyme-catalyzed reactions. The effect of elevated hydrostatic pressure (up to 0.8 kbar) on the reaction of butyrylcholinesterase with N-methyl-(7-dimethylcarbamoxy)quinolinium was determined under single-turnover conditions at 35 degrees C. The rate of carbamylation was monitored as the accumulation of the fluorescent ion, N-methyl-7-hydroxyquinolinium, in a high-pressure stopped-flow apparatus designed for the assay of fluorescence. Elevated pressure favored formation of the enzyme-substrate complex but inhibited carbamylation of the enzyme. Because a single reaction step was recorded, it was possible to interpret the data obtained under high pressure in the form of Michaelis-Menten equations. From the pressure dependence of the dissociation constant for the enzyme-substrate complex and the rate constant for carbamylation, maximal volume changes accompanying these events were determined. The value for the binding process, delta Vb = -129 ml.mol-1, is too large to be related only to volumetric changes in the active center. Substrate-induced conformational change and change of water structure appear to be the dominant contributions to the overall volume change associated with substrate binding. The large positive activation volume measured (delta V not equal to = 119 ml.mol-1) may also reflect extended structural and hydration changes. At pressures greater than 0.4 kbar, an additional pressure effect, dependent on substrate concentration, occurred in a narrow pressure interval. This effect may have resulted from a substrate-induced pressure-sensitive enzyme conformational state.     

Parameters of enzyme reactions in microcirculation

It has been shown that time-dependent change in the concentration of enzymic reaction substrate in a microcirculatory channel cell, as well as its steady spatial distribution contain information both about the structure of the microcirculatory channel and about the reaction kinetic parameters. In terms of the hypothesis about the stationary state of the enzyme-substrate complex at selective values of hydrodynamic parameters of the substrate, enzyme, channel correlation between the change in the substrate concentration, its stationary distribution, kinetic parameters and the microcirculation cell structure were estimated.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentK _m andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn²⁺ vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 μmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK _a’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE. This work was financed in part by a grant made by theUS Agency for International Development under PSTC proposal 6-163,USAID grant no. 9365542-G-00-89-42-00, and PAEC.     

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N.

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.     

Multiple conformational changes in enzyme catalysis

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

Structural and functional importance of first-shell metal ligands in the binuclear manganese cluster of arginase I

Arginase is a binuclear manganese metalloenzyme that hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of D128E, D128N, D232A, D232C, D234E, H101N, and H101E arginases I have been determined by X-ray crystallographic methods to elucidate the roles of the first-shell metal ligands in the stability and catalytic activity of the enzyme. This work represents the first structure-based dissection of the binuclear manganese cluster using site-directed mutagenesis and X-ray crystallography. Substitution of the metal ligands compromises the catalytic activity of the enzyme, either by the loss or disruption of the metal cluster or the nucleophilic metal-bridging hydroxide ion. However, the substitution of the metal ligands or the reduction of Mn(2+)(A) or Mn(2+)(B) occupancy does not compromise enzyme-substrate affinity as reflected by K(M), which remains relatively invariant across this series of arginase variants. This implicates a nonmetal binding site for substrate l-arginine in the precatalytic Michaelis complex, as proposed based on analysis of the native enzyme structure (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W. (1996) Nature 383, 554-557).     

Conformational Dynamics of Human AP Endonuclease in Base Excision and Nucleotide Incision Repair Pathways

Abstract

APE1 is a multifunctional enzyme that plays a central role in base excision repair (BER) of DNA. APE1 is also involved in the alternative nucleotide incision repair (NIR) pathway. We present an analysis of conformational dynamics and kinetic mechanisms of the full-length APE1 and truncated NΔA61-APE1 lacking the N-terminal 61 amino acids (REF1 domain) in BER and NIR pathways. The action of both enzyme forms were described by identical kinetic schemes, containing four stages corresponding to formation of the initial enzyme-substrate complex and isomerization of this complex; when a damaged substrate was present, these stages were followed by an irreversible catalytic stage resulting in the formation of the enzyme-product complex and the equilibrium stage of product release. For the first time we showed, that upon binding AP-containing DNA, the APE1 structure underwent conformational changes before the chemical cleavage step. Under BER conditions, the REF1 domain of APE1 influenced the stability of both the enzyme-substrate and enzyme-product complexes, as well as the isomerization rate, but did not affect the rates of initial complex formation or catalysis. Under NIR conditions, the REF1 domain affected both the rate of formation and the stability of the initial complex. In comparison with the full-length protein, NΔA61-APE1 did not display a decrease in NIR activity with a dihydrouracil-containing substrate. BER conditions decrease the rate of catalysis and strongly inhibit the rate of isomerization step for the NIR substrates. Under NIR conditions AP-endonuclease activity is still very efficient.     

Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity

The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.