A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.     

Hydroxylating activity of frog epidermis tyrosinase.

Trypsin activated in a similar way both the tyrosine hydroxylase and the dopa-oxidasa activities of frog epidermis tyrosinase. Several electron donors reduced or eliminated the lag period for the hydroxylating enzyme. 4 x 10(-5) M dopa was particularly effective, but without affecting the stationary activity after lag period. Tyrosine hydroxylase had KM = 2.6 X 10(-3) M for tyrosine and 2 x 10(-3) M dopa was a competitive inhibitor with Ki = 5 x 10(-4) M. The enzyme was inactivated during its actuation. Data on thermal denaturation were similar to other obtained from dopa oxidase. Our results tend to confirm our previous hypothesis that the activatory process of the enzyme is accompanied by a spatial unfolding of the enzyme molecule.     

Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8

Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid. The thermostable enzyme thus produced was extracted from the E. coli cells, purified, and crystallized. The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x 10(4). The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M and that for NAD 6.3 x 10(-4) M. The optimum pH at 75 degrees C in the presence of 1.2 M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl. The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein. Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed.     

Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI.     

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus).

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.     

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

Physico-chemical properties of molten dimer ascorbate oxidase

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.     

Effect of long wave u.v. irradiation on the affinity of haemoglobin for oxygen

Changes in the slope of haemoglobin-oxygen dissociation curve and its position were studied before and after the influence of long wave u.v. irradiation. Haemoglobin showed a lower than normal affinity for oxygen when exposed to 5.45 x 10(-3) J/cm2 and to lesser extent to doses of 10.90 x 10(-3) J/cm2. The elevation in P50 (representing PO2 at which Hb is half saturated) at these doses is mainly due to the new acidic groups which, by unfolding of this globular protein, become exposed in its surface. The fall in P50 at relatively high doses was found as a result of methaemoglobin increase and the partial dissociation of Hb tetramer to dimer and monomer.     

Interaction of RNase A with VO3- and VO2+ ions. Metal ion binding mode and protein secondary structure

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.     

Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI.     

Substrate and dilution effects on the inhibition of acetylcholinesterase by carbamates

1. The kinetics of acetylcholinesterase (EC 3.1.1.7) activity and its inhibition by eserine or by Sevin (1-naphthyl N-methylcarbamate) have been studied over the substrate concentration range 5x10(-8) to 2.5x10(-2)m. 2. Equations are given for inhibition as a function of time, substrate and inhibitor concentrations, and the relevant parameters determined at 25 degrees and 37 degrees . 3. The observed and calculated effects of time, dilution, substrate addition and enzyme concentration were in good agreement and consistent with a steady-state carbamylation by eserine or by Sevin in the presence of excess of inhibitor. 4. The quantitative destruction of either inhibitor at high enzyme concentrations implied by the carbamylation hypothesis has been confirmed experimentally. 5. The importance and possibility of allowing quantitatively for dilution and substrate effects when estimating carbamate inhibition are demonstrated.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

Partial characterization of soluble esterase from Heterodera glycines and inhibition by aldicarb and phenamiphos.

1. Homogenates of tissues from females of the nematode Heterodera glycines were clarified by centrifugation and used to initiate characterization of soluble esterases using p-nitrophenyl acetate as the substrate. 2. Optimum temperature and pH were 40 degrees C and 7.2 respectively. 3. Acetazolamide (a carbonic anhydrase inhibitor) at 10(-3) M did not inhibit enzyme activity, indicating that carbonic anhydrase was not present. 4. Phenamiphos (an organophosphate) at 10(-6) M reduced activity by 38%, whereas eserine hemisulfate (a cholinesterase inhibitor) and aldicarb (a carbamate) were not inhibitory at that concentration, indicating that there was no cholinesterase activity. 5. Eserine hemisulfate, aldicarb, and phenamiphos inhibited enzyme activity by 50% (I50) at 5 x 10(-3) M, 7.5 x 10(-4) M, and 6 x 10(-6) M, respectively. 6. Approximately 25% of the activity detected appeared due to A- and/or C-esterases. 7. The data demonstrated that aldicarb and phenamiphos were active against esterases other than acetylcholinesterase.     

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.     

Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase

The conditions in which protein stability is biologically or industrially relevant frequently differ from those in which reversible denaturation is studied. The trimeric tailspike endorhamnosidase of phage P22 is a viral structural protein which exhibits high stability to heat, proteases, and detergents under a range of environmental conditions. Its intracellular folding pathway includes monomeric and trimeric folding intermediates and has been the subject of detailed genetic analysis. To understand the basis of tailspike thermostability, we have examined the kinetics of thermal and detergent unfolding. During thermal unfolding of the tailspike, a metastable unfolding intermediate accumulates which can be trapped in the cold or in the presence of SDS. This species is still trimeric, but has lost the ability to bind to virus capsids and, unlike the native trimer, is partially susceptible to protease digestion. Its N-terminal regions, containing about 110 residues, are unfolded whereas the central regions and the C-termini of the polypeptide chains are still in the folded state. Thus, the initiation step in thermal denaturation is the unfolding of the N-termini, but melting of the intermediate represents a second kinetic barrier in the denaturation process. This two-step unfolding is unusually slow at elevated temperature; for instance, in 2% SDS at 65 degrees C, the unfolding rate constant is 1.1 x 10(-3) s-1 for the transition from the native to the unfolding intermediate and 4.0 x 10(-5) s-1 for the transition from the intermediate to the unfolded chains. The sequential unfolding pathway explains the insensitivity of the apparent Tm to the presence of temperature-sensitive folding mutations [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol. Chem. 264, 10693-10698] which are located in the central region of the chain. The metastable unfolding intermediate has not been detected in the forward folding pathway occurring at lower temperatures. The early stage of the high-temperature thermal unfolding pathway is not the reverse of the late stage of the low-temperature folding pathway.     

The effect of core destabilization on the mechanical resistance of I27

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.     

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.     

Human pyridoxal kinase: overexpression and properties of the recombinant enzyme

Pyridoxal kinase catalyses the phosphorylation of the vitamin B6. A human brain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine pyridoxal kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable information for further biochemical studies on this enzyme.