Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.     

Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes

The monomeric form of rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) was covalently coupled to CNBr-activated Sepharose and the catalytic properties of the covalently coupled monomer subunit were examined. The immobilized subunit showed one pH optimum near pH 5.6 which appears to be the characteristic pH optimum of the monomer. The enzyme-Sepharose complex exhibited the characteristic anomalous kinetic behavior at pH 5.5 but there was no turnover-induced inactivation of the immobilized enzyme at pH 4.5. The covalently coupled subunit column was examined for its ability to act as a subunit affinity chromatography medium. It was found that dissolved aryl sulfatase A was removed from solution at pH 4.5 and pH 5.0, I = 0.2, and became associated with the affinity column of Sepharose-aryl sulfatase A. The retained subunit of the enzyme could subsequently be quantitatively eluted with 0.2 m Tris-HCl, pH 7.5. Extraneous protein such as bovine serum albumin did not measureably affect the rate or equilibrium for association of the enzyme to the covalently bound subunit. The extent of binding of the enzyme to the affinity column was found to be strongly dependent on the time of equilibration and on the pH. About 90% of the enzyme was retained after 24 h at pH 5.0, I = 0.2. Under otherwise comparable conditions, use of Sepharose-6MB resulted in slightly faster association than did Sepharose-4B. Under the experimental conditions employed, the total capacity of the affinity column was approx 50% of the total aryl sulfatase A coupled to the Sepharose. The rabbit liver subunit column also permits the purification of several other aryl sulfatase A enzymes. Thus, the subunit affinity column provides a simple, convenient, and rapid procedure for the isolation of most mammalian aryl sulfatase A enzymes as well as for studying inter- and intraspecific subunit association interactions.     

The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.     

Evidence of an essential histidine residue in rabbit liver aryl sulfatase A.

A detailed study of the pH dependence of the Michaelis-Menten constants (V and K_m) of aryl sulfatase A (EC 3.1.6.1) from rabbit liver indicates that at least two functional groups (pK's ~4.3 and ~7 in the enzyme-substrate complex) participate in the enzymic degradation of substrate. Aryl sulfatase A is inactivated by diethyl pyrocarbonate (ethoxyformic anhydride). The enzyme that has been modified with this reagent can in turn be reactivated by treatment with hydroxylamine. The pH dependence of inactivation reveals a reactive group having a pK of 6.5–7.0. The results indicate that at least one histidine plays an important catalytic role in rabbit liver aryl sulfatase A, consistent with the results of earlier workers who employed diazotized sulfanilic acid. Phosphate ion, a competitive inhibitor, partially protects the enzyme from inactivation by diethyl pyrocarbonate whereas sulfate ion, also a competitive inhibitor, increases the rate of inactivation by diethyl pyrocarbonate. This result is of particular significance in view of the anomalous kinetics of aryl sulfatase A. The kinetic effects of even small amounts of sulfate ion impurities in many commercial sulfate ester substrate preparations is also discussed.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4)

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K _d) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K _d values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k _cat values than that of WT (13.4 s⁻¹). The K _d values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k _cat values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k _cat values of these interface mutants decreased as the K _d values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.     

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5-7.5

The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5–7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k_off) for βLg dimer dissociation. At pH 2.5 k_off is low (0.008 and 0.009 s⁻¹), but at higher pH (6.5 and 7.5) k_off is considerably greater (>0.1 s⁻¹). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K_D^2-1 fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The K_D^2-1 increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.     

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Ų. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.     

Dependence of reactivity and cooperativity in normal human erythrocyte glucose-6-phosphate dehydrogenase on ionic strength,pH, and temperature

The steady-state kinetics of the binding reaction of NADP⁺ to normal human erythrocyte glucose-6-phosphate dehydrogenase were studied as a function of pH, ionic strength, and temperature. The interaction coefficient obtained according to the Hill equation increases with increase in pH, ionic strength, and temperature. The observed variation of cooperative interaction is interpreted in terms of an increase in the percentage of the dimer as these environmental parameters increase. Activation energy decreases with increase in pH, the activation energy at the lowest pH being almost halved at the most alkaline pH. This behavior is explained in terms of a differential reactivity between the dimeric and tetrameric forms of the enzyme. The observation of cooperative and more reactive dimer is postulated to be a regulatory mechanism by the cell for shifting the equilibrium from one quaternary structure to the other depending on the need for NADPH.     

Covalent modification as the cause of the anomalous kinetics of aryl sulfatase A.

Mammalian aryl sulfatase A enzymes are known to exhibit an anomalous kinetic behavior in which the enzyme becomes inactivated as it catalyzes the hydrolysis of substrate. Part of the activity of this inactive, turnover-modified form of the enzyme can apparently be restored by the simultaneous presence of substrate and sulfate ion. The present experiments, conducted with 2-hydroxy-5-nitrophenyl [³⁵S]sulfate (nitrocatechol sulfate), establish that the turnover-modified enzyme is covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of ³⁵S per mole of enzyme monomer (each monomer of rabbit liver aryl sulfatase consists of two equivalent subunits). It is also shown that isolated, turnover-modified enzyme has lost 80% of its secondary structure when compared to the native enzyme. A commonly used sulfating agent, pyridine-sulfur trioxide complex brings about a similar loss of activity and of secondary structure.     

A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.     

Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent

The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted [Saier, M.H. (1980) J. Supramol. Struct. 14, 281-294]. The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed. Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions. The dimer is favored over the monomer at high ionic strength and basic pH. Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization. A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer. Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions. The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3. The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed. The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction.     

Complexes of Tungsten(VI) with Mucic Acid: a Spectrophotometric and Polarimetric Study in Aqueous Solution

Spectrophotometric work on mucic acid-W(VI) system shows the formation of three different oxoanion complexes in aqueous solution; their stability is dependent upon pH. One of the complexes is monomeric tungstodimucate and the other two are 2/2 species. An anomalous cryoscopic behaviour, similar to that of W(VI) tartaric system, has been observed for the dimeric complex formed at higher pH. The stoichiometries and conditional dissociation constants have been polarimetrically determined by means of competitive reactions between the mucic and tartaric ligands. ¹H and ¹³C NMR spectra have been interpreted for the similar complex species of both mucic and tartaric acids.     

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.     

Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glycosides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous according to electrophoresis and has M 45 ± 1 kDa. The semiinactivation time of the enzyme at 60°C is 20 min, and its K _m value for the hydrolysis of p-nitrophenyl sulfate is 8.7 ± 1 mM. It was shown that natural sulfated polyhydroxysteroids inhibit activity of the sulfatase; their I ₅₀ values depend on their structures and are within the range from 10^?3 to 10^?5 M.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Thermal,Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol⁻¹ and 14.90 Kcal mol⁻¹ for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔC_p) is 3.42 Kcal mol⁻¹ K⁻¹ for dimer. The small ΔC_p for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.     

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K_m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.     

Kinetics of reconstitution of porcine muscle lactic dehydrogenase after reversible high pressure dissociation

Porcine muscle lactic dehydrogenase can be reversibly dissociated into monomers at high hydrostatic pressure. The rate of dissociation depends on the conditions of the solvent (Schade et al., 1980, Biochemistry, in press). Maximum yields of reactivation are achieved after dissociation by 20 min incubation in 0.2 M Tris/HCl buffer or 0.2 M KCl at pH 7.6, in the presence of 10 mM dithioerylhritol and 1 mM EDTA, provided that both dissociation and reassociation are performed under anaerobic conditions. At enzyme concentrations of the order of 1 μM reactivation amounts to 9?5%/, the product of reac- tivation being indistinguishable from the enzyme in its initial native state. Based on the long-term stability of the enzyme under the optimum given conditions of reactivation, the kinetics of reconstitution after pressure release were investigated over a wide range of enzyme concentrations (1 nM < c < 1 μM). The weakly sigmoidal kinetics may be described by an irre- versible uni-bimolecular reaction scheme, corresponding to a sequential transconformation-association process. Assuming the protomers to be enzymatically inactive, the kinetic profiles may be fitted by one set of kinetic constants: k_uni = 1.5 × 10^?2 s^?1and k_bi = 7 × 10³ s^?1 M^?1, the association step belonging to either dimer or tetramer formation.