Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase

Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.     

Linkage of subunit interactions, structural changes, and energetics of coenzyme binding in tryptophan synthase

The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods. At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps. Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs. time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain. The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation. The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions. Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs. On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A

Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism.     

Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated beta 2 dimer of tryptophan synthase bienzyme complex from Escherichia coli

High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affects the structural stability, as well as the kinetics of dissociation and deactivation. The apo beta 2 dimer is deactivated faster than the holoenzyme by a factor of 10. The midpoints of the corresponding equilibrium transition curves are observed at 690 and 870 bar, respectively. As shown by hybridization of native and chemically modified beta chains, the loss of enzymatic activity is accompanied by subunit dissociation. An additional deactivating effect is produced by the pressure-induced release of the cofactor from the holoenzyme. Renaturation after decompression has been monitored by circular dichroism and intrinsic fluorescence emission. Alterations of the dichroic absorption at 222 nm reflect the recovery of the native secondary structure, while tryptophan fluorescence represents a specific probe for the native tertiary structure in the immediate neighborhood of the active center of the enzyme. By application of both methods to monitor the reconstitution of the apo beta 2 dimer, two first-order processes may be separated along the time scale. The faster phase (k1 = 1.2 X 10(-2) s-1) yields a "structured monomer" with 85% native secondary structure and the tryptophan side chain buried in its native hydrophobic environment. As indicated by sodium borohydride reduction, this intermediate is able to interact with the coenzyme pyridoxal 5'-phosphate in the correct way; however, it does not show enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching

The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The alpha subunit and the substrate L-serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine. The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5. Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation. As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holo alpha 2 beta 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594-6599]. The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.     

Covalent binding of an NAD analogue to liver alcohol dehydrogenase resulting in an enzyme-coenzyme complex not requiring exogenous coenzyme for activity.

1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.     

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

A low-resolution study of testicular lactate dehydrogenase using the molecular replacement technique

The orientation of the molecular 2-fold axes of mouse testicular lactate dehydrogenase (LDHase³-C₄) was determined by a rotation function search. These were subsequently identified with the P, Q, and R axes of dogfish LDHase-M₄. Since LDHase-C₄ crystallized with one molecule in a triclinic cell, the origin of the co-ordinate system was arbitrarily fixed at the molecular center. Structure factor phases were derived from an appropriately oriented dogfish apo LDHase-M₄ phasing model and combined with the observed structure amplitudes to produce a hybrid electron density map. Density points related by the molecular 222 point symmetry were averaged so as to remove the bias of the phasing model. At 7.5 Å resolution, the structure of the crystallized mouse LDHase-C₄ was found to be without coenzyme, with a conformation indistinguishable from that of dogfish apo LDHase-M₄.     

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.     

Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase

The crystal structure of the tetrameric alpha2beta2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two alpha subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the alpha subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.     

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase

The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.     

Conformation of aspartate aminotransferase in crystals

X-ray study of chicken cytosolic aspartate aminotransferase revealed conformational changes in the protein of two kinds: (1) a shift of the small domain adjacent to substrate-binding area due to interaction of the protein with two carboxyl groups of substrate and (2) a change in inclination of the coenzyme plane due to replacement of C = N bond of the coenzyme with Lys-258 by C = N bond with a substrate. An asymmetry in subunit behaviour is observed in both cases: the domain is shifted in one subunit and the coenzyme is rotated in other. Substrate-binding properties of each subunit are strictly dependent on the protein conformation in substrate-binding area.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin D9k and S100 beta.

The homodimeric S100 protein calcyclin has been studied in the apo state by two-dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence-specific 1H NMR assignments were obtained for all but one backbone and > 90% of the side-chain resonances. To our knowledge, the 2 x 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homonuclear methods. Sequential and medium-range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel beta-type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 x 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100 beta, and monomeric apo calbindin D9k has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three-dimensional structures of the calcyclin monomer subunit and calbindin D9k, which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D9k is monomeric, but full-length S100 proteins form homodimers.     

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.     

Metal ion reconstitution studies of yeast copper-zinc superoxide dismutase: the "phantom" subunit and the possible role of Lys7p

 Using a corrected molar extinction coefficient for yeast apo copper-zinc superoxide dismutase (CuZnSOD), we have confirmed that the metal binding properties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one Co²⁺ per protein dimer under the conditions in which the bovine and human CuZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties characteristic of the two Cu²⁺ plus two Co²⁺ per dimer or four Cu²⁺ per dimer metal-substituted bovine apo CuZnSOD derivatives were obtained for the yeast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu²⁺ plus one Co²⁺ per dimer or two Cu²⁺ per dimer. This half-metallated yeast CuZnSOD has been characterized by UV-visible and EPR spectroscopy as well as by native polyacrylamide gel electrophoresis. We conclude that yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be reconstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "phantom" subunit is not competent to bind metals in this fashion. Furthermore, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisiae that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn²⁺, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased propensity of the yeast protein to undergo changes that can occur in all CuZnSODs, and may have implications for amyotrophic lateral sclerosis. Received: 8 June 1998 / Accepted: 9 September 1998     

Half-site reactivity of the tyrosyl radical of ribonucleotide reductase from Escherichia coli

A C-terminally truncated form of protein B2, the homodimeric small subunit of ribonucleotide reductase from Escherichia coli, was found as the result of an apparently specific proteolysis. Truncated homodimers contain an intact binuclear iron center and a normal tyrosyl radical but have no binding capacity for the other ribonucleotide reductase subunit, protein B1, and are consequently enzymatically inactive. Heterodimers, consisting of one full-length and one truncated polypeptide, formed spontaneously during a chelation-reconstitution cycle and were easily separated from the two homodimeric variants. The heterodimeric form of B2 shows a weak interaction with the B1 subunit resulting in low enzyme activity. Using heterodimers containing deuterated tyrosine on the full-length side and protonated tyrosine on the truncated side, we could demonstrate that the tyrosyl radical was randomly generated in one or the other of the two polypeptide chains of the heterodimeric B2 subunit. The small subunit of ribonucleotide reductase thus conforms to a half-site reactivity.     

The crystal structure of yeast thiamin pyrophosphokinase.

BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)