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.     

Homotropic effects in aspartate transcarbamoylase: What happens when the enzyme binds a single molecule of the bisubstrate analog N-phosphonacetyl-l-aspartate?

The active sites of aspartate transcarbamoylase from Escherichia coli were titrated by measuring the decrease in the enzyme-catalyzed arsenolysis of N-carbamoyl-L-aspartate caused by the addition of the tight-binding inhibitor, N-phosphonacetyl-L-aspartate. Because the enzyme is a poor catalyst for this non-physiological reaction, high concentrations are required for the assays (more than 1000-fold the dissociation constant of the reversibly bound inhibitor) and, therefore, virtually all of the bisubstrate analog is bound. From the endpoint of the titration, 5.7 active sites were calculated, in excellent agreement with the number, six, based on the structure of the enzyme. Simple inhibition was observed only when the molar ratio of inhibitor to enzyme exceeded five; under these conditions, as shown in earlier physical chemical studies, the R-conformational state of the enzyme is the sole or predominant species. At low ratios of inhibitor to enzyme, the addition of inhibitor caused an increase in activity which is attributable to the conversion of the enzyme from the low-activity T-state to the much more active R-state. Comparison of the linear increase in activity as a function of inhibitor concentration at the low molar ratio (0.01, i.e. 1 inhibitor/600 active sites) with the activity lost at the high ratio provided a direct value for the mean number of active sites converted from the T-state to the R-state as a result of the binding of one bisubstrate analog to an enzyme molecule. This number was four with Mg X ATP or carbamoyl phosphate present and 4.7 for the enzyme in the presence of Mg X PPi, values approaching or identical to the theoretical maximum, 4.7, for a concerted transition with all of the active sites of the molecule changing from the T- to R-state upon the formation of a binary complex of hexameric enzyme with a single inhibitor. With the enzyme in the absence of effectors or with Mg X CTP present, the titrations showed that an average of two and one sites, respectively, of 4.7 possible, changed conformation upon ligand binding. These results were interpreted as a manifestation of an equilibrium between a sub-population of T- and R-state enzyme complexes containing one bound inhibitor molecule. The R-state species would represent 40% of the population for aspartate transcarbamoylase in the absence of extraneous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

pH-induced structural changes regulate histidine decarboxylase activity in Lactobacillus 30a

Histidine decarboxylase (HDC) from Lactobacillus 30a produces histamine that is essential to counter waste acids, and to optimize cell growth. The HDC trimer is active at low pH and inactive at neutral to alkaline pH. We have solved the X-ray structure of HDC at pH 8 and revealed the novel mechanism of pH regulation. At high pH helix B is unwound, destroying the substrate binding pocket. At acid pH the helix is stabilized, partly through protonation of Asp198 and Asp53 on either side of the molecular interface, acting as a proton trap. In contrast to hemoglobin regulation, pH has a large effect on the tertiary structure of HDC monomers and relatively little or no effect on quaternary structure.     

The allosteric regulation of pyruvate kinase

Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.     

pH dependent conformational changes in the T- and R-states of insulin in solution: circular dichroic studies in the pH range of 6 to 10.

Zinc insulin hexamer has been shown to undergo a phenol-induced T6 to R6 conformational transition in solution. Our circular dichroic (CD) studies demonstrate that insulin undergoes pH-dependent conformational changes over the pH range of 6-10 in the T-state and in the R- state. In order to determine which specific amino acid residues may be responsible for these pH-dependent changes, a series of insulin analogs were utilized. In the T-state, the pH dependent CD changes monitored in the far UV region have a pK of 8.2 and appear to be related to the titration of the A1-Gly amino group. Using the near UV CD a second pH-dependent conformational change was detected with a pK of 7.5 in the T-state. 1H N.M.R. studies suggest that B5-His may be responsible for this conformational transition. In the presence of m-cresol (R-state), the pK value was found to be 6.9. During this titration, the increased ellipticity for the R-state is diminishing as pH decreases from pH 8 to 6, and no difference in ellipticity was observed at 255 nm between T- and R-states at pH 6. Therefore, this may be due to the transition from the R back to the T-state.     

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)     

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.     

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.     

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.     

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.     

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.     

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.     

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.     

The allosteric mechanism of yeast chorismate mutase: a dynamic analysis

The effector-regulated allosteric mechanism of yeast chorismate mutase (YCM) was studied by normal mode analysis and targeted molecular dynamics. The normal mode analysis shows that the conformational change between YCM in the R state and in the T state can be represented by a relatively small number of low-frequency modes. This suggests that the transition is coded in the structure and is likely to have a low energetic barrier. Quantitative comparisons (i.e. frequencies) between the low-frequency modes of YCM with and without effectors (modeled structures) reveal that the binding of Trp increases the global flexibility, whereas Tyr decreases global flexibility. The targeted molecular dynamics simulation of substrate analog release from the YCM active site suggests that a series of residues are critical for orienting and "recruiting" the substrate. The simulation led to the switching of a series of substrate-release-coupled salt-bridge partners in the ligand-binding domain; similar changes occur in the transition between YCM R-state and T-state crystal structures. Thus, the normal mode analysis and targeted molecular dynamics results provide evidence that the effectors regulate YCM activity by influencing the global flexibility. The change in flexibility is coupled to the binding of substrate to the T state and release of the product from the R state, respectively.     

Use of silicate sol-gels to trap the R and T quaternary conformational states of pig kidney fructose-1,6-bisphosphatase.

Encapsulation of the homotetrameric pig kidney fructose-1,6-bisphosphatase (FBPase) in tetramethyl orthosilicate sol-gels was used to dramatically reduce the rate of the allosteric transition of the enzyme between the T and R allosteric states. When assayed in the absence of the allosteric inhibitor AMP, the enzyme encapsulated in the T-state exhibited little activity. The enzyme encapsulated in the R-state exhibited a 4-fold lower k(cat) and V(max) than the enzyme in solution, and the apparent K(m) for this enzyme was 350-fold higher than the corresponding value for the enzyme in solution. The [Mg(2+)](0.5) for the encapsulated enzyme was only 0.1 mM, compared to 0.54 mM for the normal enzyme. Magnesium activation, under both sets of conditions, was cooperative with a Hill coefficient of approximately 2. The activity of enzyme encapsulated in the R-state decreased to about 70% of initial activity within 1 min of adding AMP, it then decreased slowly to about 40% of initial activity over the following 7 h. Under the conditions tested, the encapsulated enzyme never became completely inactivated and AMP inhibition was no longer cooperative. For enzyme encapsulated in the T-state, activity was restored over approximately 7 h after removal of the AMP. The biphasic and slow responses to changing AMP levels suggest that encapsulated enzyme can be used to study the effects of local conformational changes distinct from the global quaternary conformational changes by slowing down the ability of the enzyme to carry out global rotations. The response to AMP exhibited by the encapsulated enzyme is consistent with the ability of AMP, at least partially, to directly influence the activity of the active site within each subunit.     

Structure of a single amino acid mutant of aspartate carbamoyltransferase at 2.5-A resolution: implications for the cooperative mechanism

One of the many interactions important for stabilizing the T state of aspartate carbamoyltransferase occurs between residues Tyr240 and Asp271 within one catalytic chain. The functional importance of this polar interaction was documented by site-directed mutagenesis in which the tyrosine was replaced by a phenylalanine [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. In the Tyr240----Phe mutant, the aspartate concentration required to achieve half-maximum velocity is reduced to 4.7 from 11.9 mM for the native enzyme. Here, we report an X-ray crystallographic study of the Tyr240----Phe enzyme at 2.5-A resolution. While employing crystallization conditions identical with those used to grow cytidine triphosphate ligated T-state crystals of the native enzyme, we obtain crystals of the mutant enzyme that are isomorphous to those of the native enzyme. Refinement of the mutant structure to an R factor of 0.219 (only eight solvent molecules included) and subsequent comparison to the native T-state structure indicate that the quaternary, tertiary, and secondary structures of the mutant are similar to those for the native T-state enzyme. However, the conformation of Phe240 in one of the two crystallographically independent catalytic chains contained in the asymmetric unit is significantly different from the conformation of Tyr240 in the native T-state enzyme and similar to the conformation of Tyr240 as determined from the R-state structure [Ke, H.-M., Lipscomb, W. N., Cho, Y. J., & Honzatko, R. B. (1988) J. Mol. Biol. (in press)], thereby indicating that the mutant has made a conformational change toward the R state, localized at the site of the mutation in one of the catalytic chains.     

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.