Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-Transverse Relaxation-Optimized Spectroscopy Nuclear Magnetic Resonance.

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P-bound and PEP-bound complexes on one side and the apo form and ternary complex on the other. Methyl-transverse relaxation-optimized spectroscopy (Me-TROSY) nuclear magnetic resonance was employed to gain structural information about BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly labeled with 15N and 2H and specifically labeled with δ-[13CH3]-isoleucine utilizing an isotopically labeled α-keto acid isoleucine precursor. Me-TROSY experiments were conducted on all four ligation states, and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well-resolved in chemical shift correlation maps of 13C and 1H. Assignments for 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15N,2H];Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, performed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.

The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus.

The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP) for phosphofructokinase-1 (PFK) from the moderate thermophile, Bacillus stearothermophilus (BsPFK), results from compensating enthalpy and entropy components. In BsPFK the positive coupling free energy that defines inhibition is opposite in sign from the negative enthalpy term and is therefore determined by the larger absolute value of the negative entropy term. Variants of BsPFK were made to determine the effect of adding small cavities to the structure on the allosteric function of the enzyme. The BsPFK Ile → Val (cavity containing) mutants have varied values for the coupling free energy between PEP and Fru-6-P, indicating that the modifications altered the effectiveness of PEP as an inhibitor. Notably, the mutation I153V had a substantial positive impact on the magnitude of inhibition by PEP. Van't Hoff analysis determined that this is the result of decreased entropy-enthalpy compensation with a larger change in the enthalpy term compared to the entropy term.

Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.

The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol(-1) (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol(-1) increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.

Application of three-photon excitation FCS to the study of protein oligomerization.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log-log plot of intensity against laser power.

Allosteric regulation in phosphofructokinase from the extreme thermophile Thermus thermophilus.

An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25 °C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to establish rigorously that PEP only inhibits by antagonizing the binding of Fru-6-P and not by influencing turnover, a conclusion that requires kcat to be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from Bacillus stearothermophilus but not for PFK from Escherichia coli , supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as that obtained from X-ray crystallography.

Redefining the role of the quaternary shift in Bacillus stearothermophilus phosphofructokinase.

Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose 6-phosphate (Fru-6-P), binds along the other dimer-dimer interface. Evans et al. observed that the structure with inhibitor (phosphoglycolate) bound, compared to the structure of wild-type BsPFK with substrate and activator bound, exhibits a 7° rotation about the substrate-binding interface, termed the quaternary shift [Schirmer, T., and Evans, P. R. (1990) Nature 343, 140-145]. We report that the variant D12A BsPFK exhibits a 100-fold increase in its binding affinity for PEP, a 50-fold decrease in its binding affinity for Fru-6-P, but an inhibitory coupling comparable to that of the wild type. Crystal structures of the apo and PEP-bound forms of D12A BsPFK have been determined (Protein Data Bank entries 4I36 and 4I7E , respectively), and both indicate a shifted structure similar to the inhibitor-bound structure of the wild type. D12 does not directly bind to either substrate or inhibitor and is located along the substrate-binding interface. A conserved hydrogen bond between D12 and T156 forms across the substrate-binding subunit-subunit interface in the substrate-bound form of BsPFK. The variant T156A BsPFK, when compared to the wild type, shows a 30-fold increase in PEP binding affinity, a 17-fold decrease in Fru-6-P binding affinity, and an estimated coupling that is also approximately equal to that of the wild type. In addition, the T156A BsPFK crystal structure bound to PEP is reported (Protein Data Bank entry 4I4I ), and it exhibits a shifted structure similar to that of D12A BsPFK and the inhibitor-bound structure of the wild type. The results suggest that the main role of the quaternary shift may be to influence ligand binding and not to cause the heterotropic allosteric inhibition per se.

Structure of the apo form of Bacillus stearothermophilus phosphofructokinase.

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 Å resolution (Protein Data Bank entry 3U39 ). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

An in vivo approach to isolating allosteric pathways using hybrid multimeric proteins.

Hybrid tetramers of Escherichia coli phosphofructokinase (EC 2.7.1.11; EcPFK) have been used to dissect the complicated allosteric interactions within the native tetramer. The method used previously to generate hybrids in vitro involves dissociation of the parent proteins with KSCN followed by re-association as KSCN is removed via dialysis. However, this procedure is time consuming and is plagued with low hybrid yields. Consequently, we have attempted to produce hybrids more quickly and with potentially higher yields in vivo by co-expressing the parental EcPFK protein in E. coli. Wild-type EcPFK gene was cloned into pALTER-Ex2 and pALTER-1, respectively. Site-directed mutagenesis was performed to make mutant EcPFK gene in pALTER-1. Since each vector has a different origin of replication and antibiotic selection marker, we were able to co-transform both plasmids to competent E. coli cells. Following an affinity purification column, anion-exchange chromatography was used to separate the five hybrid species (4:0, 3:1, 2:2, 1:3, 0:4). While all five hybrid species were obtained, the amount 1:3 and 0:4 hybrids were very small. By changing the expression vector for the mutant EcPFK protein from pALTER-1 to pALTER-Ex1 and the charge-tag mutations from K2E/K3E to K90E/K91E, the yield of 1:3 hybrid was substantially increased. The in vivo method does increase the yield of the hybrids produced while decreasing the time required for their isolation.

Pb2+ as modulator of protein-membrane interactions.

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.

Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.

The N-terminus of glycogen phosphorylase b is not required for activation by adenosine 5'-monophosphate.

The, so far unsuccessful, search for selective effective inhibitors of glycogen phosphorylase for the treatment of type II diabetes has made phosphorylase an active target of research for the past 20 years. Many crystallographic structures of phosphorylase are currently available to aid in this research. However, those structures have been interpreted, at least in part, on the basis of work conducted with a proteolytically derived form of phosphorylase that lacked the N-terminus (phosphorylase b'). It has been reported that phosphorylase b' shows no allostery, neither homotropic nor heterotropic. The original report on phosphorylase b' examined the allosteric characteristics over very narrow ranges of effector and substrate concentrations and reported the presence of proteolytic cleavages in addition to the removal of the N-terminus. We have applied molecular biological techniques to generate a truncate lacking the N-terminus with known primary structure, and we have established conditions for fully quantifying the allosteric effect of AMP on glycogen phosphorylase b. We report here for the first time the full thermodynamic effect of AMP on phosphorylase b. Our findings with a truncate lacking the N-terminus show that the effect of AMP binding does not depend on the N-terminus.

Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition in phosphofructokinase from Escherichia coli.

This study quantifies the contribution of each of the four unique inhibiting heterotropic interactions between the allosteric inhibitor, phosphoenolpyruvate (PEP), and the substrate, fructose 6-phosphate (Fru-6-P), in phosphofructokinase from Escherichia coli (EcPFK). The unique heterotropic interactions, previously labeled by the distances between ligand binding sites, were isolated independently by constructing hybrid tetramers. Of the four unique heterotropic PEP-Fru-6-P interactions, the 45 A interaction contributed 25%, the 30 A interaction contributed 31%, and the 23 A interaction contributed 42% of the total PEP inhibition. The 33 A interaction actually causes a small activation of Fru-6-P binding by PEP and therefore contributed -8% of the total observed PEP inhibition. The pattern of relative contribution to PEP inhibition from each interaction in EcPFK does not follow the same pattern seen in MgADP activation of EcPFK. This observation supports the conclusion that although PEP and MgADP bind to the same site, they do not use the same communication pathways to influence the active site. The pattern of relative contribution describing PEP inhibition observed in this study also does not follow the pattern determined for PEP inhibition in phosphofructokinase from Bacillus stearothermophilus, suggesting that these two highly homologous isoforms are not inhibited in the same manner by PEP.

Impact of hapten presentation on antibody binding at lipid membrane interfaces.

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K(D)) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K(D)(bulk) = 1.7 +/- 0.2 nM vs. 2.9 +/- 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K(D) = 3.6 +/- 1.1 nM vs. 2.0 +/- 0.2 microM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands' relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K(D) value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 +/- 1.1 microM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K(D) = 21 +/- 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.

Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa.

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, lambda(max), does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at lambda(max), I(F), differs significantly for the five Trp containing variants of RNase Sa; 3), the I(F) differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I(F) differences in the peptides are also present in the proteins; 4) the I(F) values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.

Peptide sequence and conformation strongly influence tryptophan fluorescence.

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.

IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site.

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.

Resolving the fluorescence response of Escherichia coli carbamoyl phosphate synthetase: mapping intra- and intersubunit conformational changes.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induces fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis.

Effects of protein-ligand associations on the subunit interactions of phosphofructokinase from B. stearothermophilus.

Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.

Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy.

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.

Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources.

As the key obligatory step in the glycolytic pathway, the regulation of phosphofructokinase (PFK-1) has been the focus of study of several laboratories. While standard cloning procedures have opened the door to study PFK from a vast array of sources, a good pfk knockout Escherichia coli strain has not previously been developed. Many laboratories rely on DF1020 or similar derivatives for PFK expression. Unfortunately, DF1020 grows poorly and does not have an inherent means for controlling expression of genes from plasmids. More importantly, however, DF1020 has a tendency to grow on minimal media when glucose is used as the sole carbon source. In this study, a new E. coli PFK expression strain lacking both PFK-1 and PFK-2 has been engineered using lambda-red mediated chromosomal deletion. The resulting strain has been designated RL257. In addition to having both pfkA and pfkB deleted, RL257 contains the lacI(q) allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.