R-state AMP complex reveals initial steps of the quaternary transition of fructose-1,6-bisphosphatase

AMP transforms fructose-1,6-bisphosphatase from its active R-state to its inactive T-state; however, the mechanism of that transformation is poorly understood. The mutation of Ala(54) to leucine destabilizes the T-state of fructose-1,6-bisphosphatase. The mutant enzyme retains wild-type levels of activity, but the concentration of AMP that causes 50% inhibition increases 50-fold. In the absence of AMP, the Leu(54) enzyme adopts an R-state conformation nearly identical to that of the wild-type enzyme. The mutant enzyme, however, grows in two crystal forms in the presence of saturating AMP. In one form, the AMP-bound tetramer is in a T-like conformation, whereas in the other form, the AMP-bound tetramer is in a R-like conformation. The latter reveals conformational changes in two helices due to the binding of AMP. Helix H1 moves toward the center of the tetramer and displaces Ile(10) from a hydrophobic pocket. The displacement of Ile(10) exposes a hydrophobic surface critical to interactions that stabilize the T-state. Helix H2 moves away from the center of the tetramer, breaking hydrogen bonds with a buried loop (residues 187-195) in an adjacent subunit. The same hydrogen bonds reform but only after the quaternary transition to the T-state. Proposed here is a model that accounts for the quaternary transition and cooperativity in the inhibition of catalysis by AMP.     

Tryptophan fluorescence reveals the conformational state of a dynamic loop in recombinant porcine fructose-1,6-bisphosphatase

Wild-type porcine fructose-1,6-bisphosphatase (FBPase) has no tryptophan residues. Hence, the mutation of Try57 to tryptophan places a unique fluorescent probe in the structural element (loop 52-72) putatively responsible for allosteric regulation of catalysis. On the basis of steady-state kinetics, circular dichroism spectroscopy, and X-ray crystallography, the mutation has little effect on the functional and structural properties of the enzyme. Fluorescence intensity from the Trp57 mutant is maximal in the presence of divalent cations, fructose 6-phosphate and orthophosphate, which together stabilize an R-state conformation in which loop 52-72 is engaged with the active site. The level of fluorescence emission decreases monotonically with increasing levels of AMP, an allosteric inhibitor, which promotes the T-state, disengaged-loop conformation. The titration of various metal-product complexes of the Trp57 mutant with fructose 2,6-bisphosphate (F26P(2)) causes similar decreases in fluorescence, suggesting that F26P(2) and AMP individually induce similar conformational states in FBPase. Fluorescence spectra, however, are sensitive to the type of divalent cation (Zn(2+), Mn(2+), or Mg(2+)) and suggest conformations in addition to the R-state, loop-engaged and T-state, loop-disengaged forms of FBPase. The work presented here demonstrates the utility of fluorescence spectroscopy in probing the conformational dynamics of FBPase.     

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.     

Structures of mammalian and bacterial fructose-1,6-bisphosphatase reveal the basis for synergism in AMP/fructose 2,6-bisphosphate inhibition

Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.     

Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH

The T----R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis. To promote the T----R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively. In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM. These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition. We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4 A). The R-state structure in which the T----R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for the three structures.     

Analysis of the ligand-promoted global conformational change in aspartate transcarbamoylase. Evidence for a two-state transition from boundary spreading in sedimentation velocity experiments

A global conformational change in the regulatory enzyme aspartate transcarbamoylase of Escherichia coli was demonstrated 20 years ago by the 3.5% decrease in the sedimentation coefficient of the enzyme upon its interaction with carbamoyl phosphate and saturating amounts of the aspartate analog succinate. This "swelling" of aspartate transcarbamoylase attributable to the T----R allosteric transition was observed also in subsequent studies when the enzyme was completely saturated with the bisubstrate analog N-(phosphonacetyl)-L-aspartate. In neither of these studies was a direct attempt made by an analysis of boundary spreading (expressed as an apparent diffusion coefficient) on partially liganded enzyme to determine whether the solution contained only T and R-state molecules, as expected for a concerted transition, or a mixture of more than two distinct conformational states. The sensitivity of boundary spreading measurements was tested with a known mixture of fully liganded wild-type enzyme (R-state) and an inactive T-state mutant that did not bind either succinate or the bisubstrate ligand. This experiment yielded broad boundaries with an apparent diffusion coefficient about 10% greater than that of T-state enzyme, due to the differential sedimentation of the two independent species. Identical boundary spreading was obtained theoretically by simulating an equimolar mixture of T and R-state aspartate transcarbamoylase. These results proved that the boundary spreading measurement was sensitive to the presence of heterogeneity. Analogous experiments with only wild-type enzyme in the presence of sub-stoichiometric amounts of the tightly bound bisubstrate ligand sufficient to promote a 1.8% decrease in sedimentation coefficient also exhibited broader boundaries, corresponding to a 10% increase in the apparent diffusion coefficient relative to the unliganded enzyme. In contrast, such broad boundaries were not observed in experiments when the weakly bound succinate was present in quantities sufficient to cause the same 1.8% decrease in sedimentation coefficient. The differences in boundary spreading observed with the two active-site ligands were accounted for by the affinities of the respective ligands for the enzyme and the transport theory of a ligand-promoted isomerization of the protein. In the presence of sub-stoichiometric levels of the tight-binding bisubstrate ligand, the dynamic equilibrium between the T and the R-state is essentially uncoupled and the species sediment at slightly different rates to give broad boundaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a "latch", the beta15-alphaK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.     

Binding of AMP to two of the four subunits of pig kidney fructose-1,6-bisphosphatase induces the allosteric transition.

To study the allosteric transition in pig kidney fructose 1,6-bisphosphatase (FBPase), we constructed hybrids in which subunits have either their active or regulatory sites rendered nonfunctional by specific mutations. This was accomplished by the coexpression of the enzyme from a plasmid that contained two slightly different copies of the cDNA. To resolve and purify each of the hybrid enzymes, six aspartic acid codons were added before the termination codon of one of the cDNAs. The addition of these Asp residues to the protein did not alter the kinetic or allosteric properties of the resulting FBPase. Expression of the enzyme from a dual-gene plasmid resulted in the production of a set of five different enzymes (two homotetramers and three hybrid tetramers) that could be purified by a combination of affinity and anion-exchange chromatography because of the differential charge on each of these species. The hybrid with one subunit that only had a functional regulatory site (R) and three subunits that only had a functional active site (A) exhibited biphasic AMP inhibition. Analysis of these data suggest that the binding of AMP to the R subunit is able to globally alter the activity of the other three A subunits. The hybrid composed of two R and two A subunits is completely inhibited at an AMP concentration of approximately 0.5 mM, 100-fold less than the concentration required to fully inhibit the A(4) enzyme. The monophasic nature of this cooperative inhibition suggests that the AMP binding to the two R subunits is sufficient to completely inhibit the enzyme and suggests that the binding of AMP to only two of the four subunits of the enzyme induces the global allosteric transition from the R to the T state.     

Structure and cooperativity of a T-state mutant of histidine decarboxylase from Lactobacillus 30a

Histidine decarboxylase (HDC) from Lactobacillus 30a converts histidine to histamine, a process that enables the bacteria to maintain the optimum pH range for cell growth. HDC is regulated by pH; it is active at low pH and inactive at neutral to alkaline pH. The X-ray structure of HDC at pH 8 revealed that a helix was disordered, resulting in the disruption of the substrate-binding site. The HDC trimer has also been shown to exhibit cooperative kinetics at neutral pH, that is, histidine can trigger a T-state to R-state transition. The D53,54N mutant of HDC has an elevated Km, even at low pH, indicating that the enzyme assumes the low activity T-state. We have solved the structures of the D53,54N mutant at low pH, with and without the substrate analog histidine methyl ester (HME) bound. Structural analysis shows that the apo-D53,54N mutant is in the inactive or T-state and that binding of the substrate analog induces the enzyme to adopt the active or R-state. A mechanism for the cooperative transition is proposed.     

Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N-terminal tail occurs, while the loop of chain containing residues 192-196 and residues 43'-49' shift to accommodate the ligand. Structural comparisons show that the T-state GPa-glucose-W1807 structure is overall more similar to the T-state GPb-W1807 complex structure than to the GPa-glucose complex structure, indicating that W1807 is able to transform GPa to the T'-like state already observed with GPb. The structures provide a rational for the potency of the inhibitor and explain GPa allosteric inhibition of activity upon W1807 binding.     

Control of kinetics by cooperative interactions

Cooperative effects in ligand binding and dissociation kinetics are much less investigated than steady state kinetics or equilibrium binding. Nevertheless, cooperativity in ligand binding leads necessarily to characteristic properties with respect to kinetic properties of the system. In case of positive cooperativity as found in oxygen binding proteins, a typical property is an autocatalytic ligand dissociation behavior leading to a time dependent, apparent ligand dissociation rate. To follow systematically the influence of the various potentially involved parameters on this characteristic property, simulations based on the simple MWC model were performed which should be relevant for all types of models based on the concept of an allosteric unit. In cases where the initial conformational distribution is very much dominated by the R-state, the intrinsic kinetic properties of the T-state are of minor influence for the observed ligand dissociation rate. Even for fast conformational transition rates, the R-state properties together with the size of the allosteric unit and the allosteric equilibrium constant define the shape of the curve. In such a case, a simplified model of the MWC-scheme (the irreversible n-chain model) is a good approximation of the full scheme. However, if in the starting conformational distribution some liganded T-molecules are present (a few percent is enough), the average off-rates can be significantly altered. Thus, the assignment of the initial rates to R-state properties has to be done with great care. However, if the R-state strongly dominates initially it is even possible to get an estimation of the lower limit for the number of interacting subunits from kinetic data: similar to the Hill-coefficient for equilibrium conditions, a measure for "kinetic cooperativity" can be derived by comparing initial and final ligand dissociation rates.     

Allosteric inhibition via R-state destabilization in ATP sulfurylase from Penicillium chrysogenum

The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), was determined to 2.6 A resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 A movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition by destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.     

Allosteric control of quaternary states in E. coli aspartate transcarbamylase

Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when ATP binds. When the R-state is produced by binding of phosphonoacetamide and malonate, addition of CTP or CTP + UTP decreases the separation of catalytic trimers by 0.5 A. In the unliganded Glu239----Gln mutant, in which the T-state is destabilized so that the enzyme exists in an intermediate quaternary state, ligation of ATP transforms the mutant enzyme to the R-state, whereas CTP converts this enzyme to the T-state. Thus, this mutant is much more sensitive to heterotropic allosteric control than is the native enzyme. In this communication we propose a preliminary model based on new crystallographic results that heterotropic regulation occurs partly through control of the quaternary structure by these effectors, thus regulating catalysis.     

Ligand binding kinetic studies on the hybrid hemoglobin alpha (human):beta (carp): a hemoglobin with mixed conformations and sequential conformational changes

Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) alpha (human):beta (carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human alpha-chain is R state and the carp beta-chain is T state. We have no evidence at this pH that the carp beta-chain ever assumes the R conformation. At pH 6, the human alpha-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp beta-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human alpha-chains are in the R state, the two beta-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the beta-chains first assume T-state properties, followed by the alpha-chains. The rate of the R-T conformational change for the carp beta-chains is at least 300 times greater than that for the human alpha-chains. At pH 9, the R----T conformational transition rate is at least 200 times slower than that for human hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.     

Allosteric effectors and trehalose protect larval Manduca sexta fat body glycogen phosphorylase B against thermal denaturation

In this paper we assessed the ability of modulators of the activity of glycogen phosphorylase b from the fat body of larval Manduca sexta to stabilize the enzyme against thermal denaturation. This approach has allowed us to distinguish between modulators that stabilize the enzyme, presumably through some conformational effect, from those that do not affect thermal stability. For example, 5'-AMP and 5'-IMP are both positive modulators of the enzyme and the K(m)s for AMP and IMP were similar, 0.71 and 1.09 mM, respectively. However, the V(max) for AMP (123 nmol/mg/min) was 10 times higher than the value found for IMP (12.5 nmol/mg/min) and AMP increased the thermal stability of glycogen phosphorylase b, however IMP did not increase the enzyme's thermal stability. Indeed, IMP decreased both the allosteric activation of the enzyme by AMP and the thermal protection conferred by AMP. The allosteric inhibitors ADP and ATP, which in vertebrate phosphorylase bind to the same site as AMP, both increased the thermal stability of the enzyme, however with less efficiency than AMP. Inorganic phosphate increased thermal stability, but glycogen and amylose did not. Glycerol, at 600 mM, protected the enzyme against thermal inactivation, whereas sorbitol at the same concentration did not show any effect. Among the polyols tested, trehalose was the most effective in conferring thermal stability. In fact, in the presence of 20 mM AMP and 600 mM trehalose, 90% of the enzyme activity remained after 20 min at 60 degrees C.     

The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T<-->R equilibrium

The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues. We show here that, in the presence of the nucleotide effector ATP, known to stimulate the enzyme activity, the scattering profiles show a marked dependence on the metal bound to ATP. Whereas ATP has no major effect on the scattering pattern of ATCase, a saturating concentration of Mg-ATP notably modifies the scattering profile of the enzyme, either in the absence or in the presence of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA). The transition with PALA in the presence of this metal-nucleotide complex remains concerted. Furthermore, Mg-ATP, as already observed with ATP, has no detectable direct effect on the T to R transition. The experimental scattering curves in the presence of Mg-ATP were fitted by a modeling approach using rigid body movements of the regulatory subunits and the catalytic trimers in the crystal structures. While the differences observed in the T-state in the presence of Mg-ATP are essentially attributed to the binding per se of the nucleotide, the solution structure of the R-state complexed to Mg-ATP is even more extended along the 3-fold axis than the previously described R solution structure, which is already more stretched out along the same axis than the crystal R structure. Based on the crystal structure of the enzyme in the R-state complexed with free ATP, a proposal is made to account for the effect of magnesium.     

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.     

Crystal structure of horse carbonmonoxyhemoglobin-bezafibrate complex at 1.55-A resolution. A novel allosteric binding site in R-state hemoglobin

Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug. The high resolution crystal structure of horse carbonmonoxyhemoglobin in complex with bezafibrate reveals that the bezafibrate molecule lies near the surface of the E-helix of each alpha subunit and the complex maintains the quaternary structure of the R-state. Binding is caused by the close fit of bezafibrate into the binding pocket, which is composed of some hydrophobic residues and the heme edge, suggesting the importance of hydrophobic interactions. Upon binding of bezafibrate, the distance between Fe and the N epsilon(2) of distal His E7(alpha 58) is shortened by 0.22 A in the alpha subunit, whereas no significant structural changes are transmitted to the beta subunit. Oxygen equilibrium studies of R-state-locked hemoglobin with bezafibrate in a wet porous sol-gel indicate that bezafibrate selectively lowers the oxygen affinity of one type of subunit within the R-state, consistent with the structural data. These results disclose a new allosteric mechanism of bezafibrate and offer the first demonstration of how the allosteric effector interacts with R-state hemoglobin.     

Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5''-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.