Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 Å resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA.

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved.

Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening.

We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP). The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector. A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate. The mutant enzyme is also not activated by F1P or F6P. The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding. Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme. One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector. These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery. Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.

Sites of binding and orientation in a four-location model for protein stereospecificity.

The stereospecificity of the enzyme isocitrate dehydrogenase was examined by steady-state kinetics and x-ray crystallography. The enzyme has the intriguing property that the apoenzyme in the absence of divalent metal showed a selectivity for the inactive l-enantiomer of the substrate isocitrate, whereas the enzyme containing magnesium showed selectivity for the physiologically active d-enantiomer. The hydrogen atom on the C2 carbon that is transferred during the reaction was, in both the d- and l-isocitrate complexes, in an orientation very close to that expected for delivery of a hydride ion to the cosubstrate NADP+. The beta-carboxylate that is eliminated as a CO2 molecule during the reaction occupied the same site on the protein in both the d- and l-isocitrate complexes. In addition, the C3 carbon was in the same protein site in both the d- and l-enantiomers. Only the fourth group, the OH atom, was in a very different position in the apo enzyme and in the metal-containing complexes. A four-location model is necessary to explain the enantiomeric specificity of IDH in contrast to the conventional three-point attachment model. The thermodynamic and kinetic ramifications of this model are explored.

Characterization of novel rad6/ubc2 ubiquitin-conjugating enzyme mutants in yeast.

Null mutations in the RAD6/UBC2 gene encoding an E2 ubiquitin-conjugating enzyme cause deficiencies in DNA repair, N-end-rule protein degradation, sporulation and telomeric silencing, and alter the preferred integration positions for Ty1 retrotransposons. Here we selected for mutants of RAD6 that cause a release of telomeric silencing. Some alleles retained nearly wild-type ability for sporulation, DNA repair and the degradation of proteins. Alteration in Ty1 integration-site bias accompanied some of these alleles. The possibility that some mutations specifically affect binding of an unknown protein that works with Rad6 in its silencing role, but is not required for DNA repair or N-end-rule activity, is discussed in terms of the Rad6 crystal structure.

MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein.

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent K(d) of unphosphorylated NtrC for MgATPgammaS is 100 microM at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 microM at 37 degrees C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma(54)-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn(2+) binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.

Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis.

Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively. Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK. The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK. The detritiation of 3-[(3)H]pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively. This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M. Isotope trapping was used to characterize enolpyruvate protonation by K240M. While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M. In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8. 8 observed with wild-type YPK. The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.

Millisecond Laue structures of an enzyme-product complex using photocaged substrate analogs.

The structure of a rate-limited product complex formed during a single initial round of turnover by isocitrate dehydrogenase has been determined. Photolytic liberation of either caged substrate or caged cofactor and Laue X-ray data collection were used to visualize the complex, which has a minimum half-life of approximately 10 milliseconds. The experiment was conducted with three different photoreactive compounds, each possessing a unique mechanism leading to the formation of the enzyme-substrate (ES) complex. Photoreaction efficiency and subsequent substrate affinities and binding rates in the crystal are critical parameters for these experiments. The structure suggests that CO2 dissociation is a rapid event that may help drive product formation, and that small conformational changes may contribute to slow product release.

The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences.

Small structural perturbations in the enzyme isocitrate dehydrogenase (IDH) were made in order to evaluate the contribution of precise substrate alignment to the catalytic power of an enzyme. The reaction trajectory of IDH was modified (i) after the adenine moiety of nicotinamide adenine dinucleotide phosphate was changed to hypoxanthine (the 6-amino was changed to 6-hydroxyl), and (ii) by replacing Mg2+, which has six coordinating ligands, with Ca2+, which has eight coordinating ligands. Both changes make large (10(-3) to 10(-5)) changes in the reaction velocity but only small changes in the orientation of the substrates (both distance and angle) as revealed by cryocrystallographic trapping of active IDH complexes. The results provide evidence that orbital overlap produced by optimal orientation of reacting orbitals plays a major quantitative role in the catalytic power of enzymes.

Metal-ion-mediated allosteric triggering of yeast pyruvate kinase. 1. A multidimensional kinetic linked-function analysis.

Regulation of the glycolytic pathway is considered to be primarily achieved by the carbon metabolites resulting from glucose metabolism [e.g., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citrate] and by the ATP charge of the cell. The divalent cations (e.g., Mg2+ and Mn2+) have not been considered as having regulatory roles in glycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway. Using a kinetic linked-function analysis of steady-state kinetic data for the interactions of PEP, FDP, and Mn2+ with yeast pyruvate kinase (YPK), we have found that the divalent metal is the principal trigger of the allosteric responses observed with this enzyme. The interaction of Mn2+ to YPK enhances the interaction of FDP by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol. The simultaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energies. Surprisingly, the binding of the allosteric activator FDP does not directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands. Thus, communication between the PEP and FDP sites occurs structurally through the metal by an allosteric relay mechanism. These conclusions are supported by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK [Mesecar, A. D., & Nowak, T. (1997) Biochemistry (following paper in this series)]. Our findings raise important questions as to the possible roles of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.

Metal-ion-mediated allosteric triggering of yeast pyruvate kinase. 2. A multidimensional thermodynamic linked-function analysis.

A role has been proposed for the free divalent metal in triggering the allosteric responses of yeast pyruvate kinase based upon a kinetic linked-function analysis [Mesecar, A. D., & Nowak, T. (1997a) (preceding paper in this series)]. The major conclusion from the analysis is that the allosteric activator, fructose 1,6-diphosphate (FDP), does not directly communicate with the substrate, phosphoenolpyruvate (PEP), at the active site of the enzyme: it is Mn2+ that mediates the allosteric communication between the PEP and FDP sites in an allosteric relay mechanism. Assumptions were necessary to treat kinetic parameters as thermodynamic parameters, and the presence of the substrate ADP was necessary for the kinetic analysis. In this study, the influence of FDP on the interactions of PEP and Mn2+ and the influence of PEP and Mn2+ on the interaction of FDP with YPK were measured, where possible, by direct binding methods in the absence of ADP. Direct binding data were then subjected to a thermodynamic linked-function analysis for a heterotropic, three ligand coupled system in order to ascertain the two and three ligand coupling free energies. The two ligand coupling free energies deltaG(Mn-PEP), deltaG(Mn-FDP), and deltaG(PEP-FDP) are -3.88, -1.09, and -0.22 kcal/mol, respectively. These values indicate that positive, heterotropic interactions exist between each of these ligand pairs. The three ligand coupling free energy term, deltaG(Mn-PEP-FDP), indicates that simultaneous binding of Mn2+, PEP, and FDP is considerably favored over the sum of their independent binding free energies by -6.6 kcal/mol. These results demonstrate the key role of the metal in the modulation of ligand binding and are consistent with the values and the relationships of the kinetic parameters obtained from the kinetic linked-function analysis.