Sugar transport by the bacterial phosphotransferase system. Characterization of the sulfhydryl groups and site-specific labeling of enzyme I

Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.     

Sugar transport by the bacterial phosphotransferase system. The intrinsic fluorescence of enzyme I

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system has 2 tryptophan residues/monomer, as determined spectrophotometrically. The tryptophan fluorescence has been investigated with the aid of nanosecond time-resolved techniques. The decay of the fluorescence intensity was analyzed in terms of a biexponential function. The contribution of the emission associated with the shorter decay constant increases from 17-19% at 1 degree C to 43-44% at room temperature. Decay-associated spectra obtained with Enzyme I indicate different spectral distributions associated with the two decay constants. The measurement of tumbling of Enzyme I as a function of temperature revealed a transition of rotational rates between 5 and 15.5 degrees C. Global analysis allowed decomposition of the anisotropy decay into a formulation consistent with monomer and dimer rotational contributions.     

Proximity of sulfhydryl groups in lens proteins. Excimer fluorescence of pyrene-labeled crystallins

Lens proteins labeled with the -SH-specific reagents N-(1-pyrene)-maleimide (PM) and N-(1-pyrene)-iodo-acetamide (PIA) exhibited pyrene excimer fluorescence around 480 nm. Among the gamma-fractions, only gamma II showed excimer band at room temperature with both probes PM and PIA. As the temperature increased, PM-labeled gamma IIIA, gamma IIIB, and gamma IV also began to exhibit excimer around 55 degrees C, which did not disappear at a very high temperature (85 degrees C). With PIA, gamma IIIA and gamma IVA did not show excimer at any temperature. The beta-crystallins, on the other hand, revealed a very strong excimer/monomer intensity ratio at room temperature, which decreased with an increase in temperature. Life-time measurements indicated a difference in the micro-environments around the labeled -SH residues. The origin of the excimer band as well as temperature effects on this band have been explained on the basis of intra- and inter-molecular interaction among the Cys residues in the lens proteins. The temperature-dependent CD studies further indicated retention of thermodynamic stability of the crystallins after chemical modifications. Both PM and PIA could be used conveniently to probe -SH proximity, determine the ease and extent of disulfide formation, and monitor the dynamics of lens protein conformation, all of which are critically important with regard to cataract formation.     

Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family

Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.     

Ordered disruption of subunit interfaces during the stepwise reversible dissociation of Escherichia coli phosphofructokinase with KSCN

The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN. At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers. These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction. Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation. The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation. This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer. The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers. The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor. Two successive processes can be observed during reassociation of the protein upon KSCN dilution. First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved intrinsic fluorescence of Enzyme I. The monomer/dimer transition.

Enzyme I of the bacterial phosphotransferase system can exist in a monomer/dimer equilibrium which may have functional significance. Each monomer contains two tryptophan residues. It is demonstrated that the decay of both the monomer and the dimer can be described by a biexponential. The decay times depend on the temperature and at 6 degrees C the decay times are tau 1 = 0.4 ns and tau 2 = 3.2 ns for the monomer and tau 3 = 3.2 ns and tau 4 = 7.2 ns for the dimer form of the enzyme. The changes in the fluorescence decay parameters can be utilized to measure the equilibrium constant for the monomer/dimer transition.     

Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism

The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.     

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.     

Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.     

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

One site mutation disrupts dimer formation in human DPP-IV proteins

DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.     

Fluorescent-labeled crosslinks in collagen: Pyrenebutyrylhydrazine

The aldehydes present in acid-soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH₄, does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin-treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α-chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β-chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides.     

Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study

The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was modified by several arginine-specific reagents. Modifications by 2,3-butanedione led to the loss of activity of the enzyme, but the binding of p-hydroxybenzoate and NADPH to the enzyme was little or not at all affected. However the formation of the enzyme-substrate complex of the modified enzyme was accompanied by an increase of the fluorescence of protein-bound FAD, in contrast to that of native enzyme which leads to quenching of the fluorescence. Enzyme modified by phenylglyoxal did not bind p-hydroxybenzoate nor NADPH. Quantification and protection experiments showed that two arginine residues are essential and a model is described which accounts for the results. Modification by 4-hydroxy-3-nitrophenylglyoxal reduced the affinity of the enzyme for the substrate and NADPH. The ligands offered no protection against inactivation. From this it is concluded that one arginine residue is essential at some stage of the catalysis. This residue is not associated with the substrate- or NADPH-binding site of the enzyme. Time-resolved fluorescence studies showed that the average fluorescence lifetime and the mobility of protein-bound FAD are affected by modification of the enzyme.     

Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

Chemical modification of SH groups of E. coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.     

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.     

Picosecond laser fluorometry of FAD of D-amino acid oxidase-benzoate complex

Formation of a complex of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, an enzyme-substrate complex model, was studied by measuring the fluorescence life-time of the coenzyme FAD of the complex by using a mode-locked Nd:YAG laser and a streak camera. The value of lifetime was 60 +/- 10 ps in the monomer of the complex and it was extremely short (much less than 5 ps) in the dimer of the complex. Since the values of fluorescence lifetime of the coenzyme are 130 ps in the monomeric form of free enzyme and 40 ps in the dimeric form of free enzyme, the decrease in the lifetime upon complex formation with benzoate is slight in the monomer (reduced to one-half) whereas marked in the dimer (reduced to less than 1/10). By analyzing the fluorescence decay curve, a dissociation constant of the monomer-dimer equilibrium of the complex was evaluated to be 0.4 +/- 0.3 microM, which is much smaller than that in free enzyme. Fluorescence analysis under steady state excitation revealed that the apparent dissociation constant (K) of FAD from the enzyme was decreased by 1:1000 upon the complex formation. Relative quantum yield of the fluorescence of FAD in the complex to that of free FAD exhibited appreciable dependence on the complex concentration: greater in the monomer and less in the dimer. These results suggest that a molecular interaction between FAD and amino acid residue(s) is strengthened by the complex formation, which contributes to a remarkable conformational change in the protein moiety of the complex.     

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.     

Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.