Kinetic and structural analysis of alpha-D-Glucose-1-phosphate cytidylyltransferase from Salmonella typhi

Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of the surface lipopolysaccharides of some pathogenic bacteria. It is synthesized via a complex biochemical pathway that is initiated by the formation of CDP-D-glucose. The production of this ligand is catalyzed by the enzyme glucose-1-phosphate cytidylyltransferase, which utilizes alpha-D-glucose 1-phosphate and MgCTP as substrates. Previous x-ray crystallographic investigations have demonstrated that the Salmonella typhi enzyme complexed with the product CDP-glucose is a fully integrated hexamer displaying 32 point group symmetry. The binding pocket for CDP-glucose is shared between two subunits. Here we describe both a detailed kinetic analysis of the cytidylyltransferase and a structural investigation of the enzyme complexed with MgCTP. These data demonstrate that the reaction catalyzed by the cytidylyltransferase proceeds via a sequential rather than a Bi Bi ping-pong mechanism as was previously reported. Additionally, the enzyme utilizes both CTP and UTP equally well as substrates. The structure of the enzyme with bound MgCTP reveals that the binding pocket for the nucleotide is contained within one subunit rather than shared between two. Key side chains involved in nucleotide binding include Thr(14), Arg(15), Lys(25), and Arg(111). In the previous structure of the enzyme complexed with CDP-glucose, those residues defined by Thr(14) to Ile(21) were disordered. The kinetic and x-ray crystallographic data presented here support a mechanism for this enzyme that is similar to that reported for the glucose-1-phosphate thymidylyltransferases.     

Structure of Panulirus interruptus hemocyanin at 5 Å resolution

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P2₁ with two molecules in the unit cell and cell dimensions $\text{a = 119.8}\text{A}\text{?}\text{, b = 193.1}\text{A}\text{?}\text{, c = 122.2}\text{A}\text{?}\text{and}\text{β = 118.1 °}$. With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis.X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis.For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å.Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer.The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.     

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.     

Crystal structure of ATP sulfurylase from Penicillium chrysogenum: insights into the allosteric regulation of sulfate assimilation

ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.     

Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli

Crystals of uridine phosphorylase from Escherichia coli K12 have been grown from solutions of polyethylene glycol 4000. The crystals are trigonal, space group R3; the hexagonal axes are a = 154.4 A and c = 49.4 A. The crystals are quite stable to x-rays and diffract beyond 2.6 A resolution. It appears that the molecule is a hexamer with a subunit molecular weight of 27,500 and utilizes the 3-fold symmetry of the space group, resulting in two subunits/asymmetric unit.     

The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications

The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.     

Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A

The principal elements of the secondary, tertiary and quaternary structure of the tetrameric human plasma prealbumin molecule have been determined by Fourier refinement of X-ray diffraction data at 1.8 Å resolution. The subunit has an extensive β-structure composed of eight strands organised into two four-stranded sheets. There is also one short α-helix. The tertiary structure is largely determined by the association of the two β-sheets. Important contributions to the tertiary structure are made by three tyrosines and one aspartic acid involved in side-chain-main-chain interactions; a buried histidine associated with a group of internal water molecules; and a compact cluster of seven aromatic residues. Quaternary interactions occur at two sets of interfaces closely organised around two of the three molecular 2-fold axes. The exclusive monomer-monomer interface is chiefly concerned with antiparallel hydrogen bond interactions which extend the two four-stranded sheets in the monomers to eight-stranded sheets in the dimer. One of the sheet interactions includes water molecules and tyrosine hydroxyls in the hydrogen bond pattern. The dimers associate through both hydrophilic and hydrophobic interactions at interfaces that involve all four monomers.     

4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy.

4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta 1, residues 2-8; beta 2, residues 39-45), a beta-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The beta 1 strand is parallel to the beta 2 strand of the same subunit on the basis of cross stand NH(i)-NH(j) NOEs in a 2D 15N-edited 1H-NOESY spectrum of hexameric 4-OT containing two 15N-labeled subunits/hexamer. The beta 1 strand is also antiparallel to another beta 1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the beta-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.     

P.versicolor龙虾D-甘油醛-3-磷酸脱氢酶的X射线晶体学研究——分子置换法结构测定

由P.versicolor龙虾尾肌提取的HOIO-D-甘油醛-3-磷酸脱氢酶(GAPDH),已长出可供Χ射线衍射用的晶体。初步Χ射线晶体学研究确定:此酶晶体属於C2空间群,不对称单位内含有半个分子,分子坐落在二重轴上。以Homarus Amercanus龙虾GAPDH结构为模型结构,应用分子置换技术进行了低分辨率Χ射线结构分析,结果表明:分子内亚基排列具有222对称性,分子Q轴平行于晶体学二重轴b,分子P和R轴分别平行于晶体学a和c轴。按分子置换法推出的结构模型算得5A分辨率的晶体学R因子为0.46。并获得了一套5A。分辨率的电子密度图。此酶的几种同晶型晶体,特别是荧光NAD衍生物晶体的较高分辨率的结构分析工作正在进行中。     

Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.     

Effect of oligomerization on the properties of essential SH-groups of mitochondrial creatine kinase

The properties of creatine kinase isolated from bovine heart mitochondria in dimeric (Mr = 84 +/- 6 kD) and octameric (Mr = 340 +/- 17 kD) forms were compared with those of the earlier described hexameric form of the enzyme (Mr = 240 +/- 12 kD). The kinetics of SH-group modification by DTNB, the inactivation kinetics as well as the number of modified SH-groups point to significant differences between the three oligomeric forms of the enzyme. Each subunit of creatine kinase was found to possess one "fast" essential cysteine residue whose modification by DTNB and iodoacetamide led to enzyme inactivation. The formation of an analog of the transition state complex (E--MgADP--NO3--creatine) was paralleled with partial protection of only the "fast" cysteine residue which manifested itself in the decrease of the rate of its interaction with DTNB in all the three oligomeric forms. Dimer association into a hexamer and octamer occurred in parallel with a decrease of the affinity of essential SH-groups of cysteine for DTNB in 50% of the oligomeric molecule subunits. Thus, in the dimer two essential SH-groups were rapidly modified by DTNB at the same rate: k1 = k2 = (23.9 +/- 5.6).10(4) M-1 min-1. Within the hexamer, the rate of modification of 3 out of 6 SH-groups was practically unchanged: k1 = (10.6 +/- 2.3).10(4) M-1 min-1. Another 3 SH-groups in the remaining 50% of the subunits were partly masked, which manifested itself in a 10-fold decrease of their modification rate: k2 = (1.12 +/- 0.28).10(4) M-1 min-1. Within the octamer, the SH-groups rapidly interacted with DTNB only on 4 subunits: k1 = (20.7 +/- 2.2).10(4) M-1 min-1, whereas in the remaining 4 octamer subunits a practically complete masking of essential SH-groups was observed, as a result of which these groups became inaccessible to DTNB. This manifested itself in a 1000-fold decrease of the rate of SH-group modification by DTNB which reached that of non-essential SH-group modification. In has been found that a complete loss of the octamer activity is due to the modification of only 4 SH-groups which interact with DTNB at a high rate. A model for subunit association into a dimer, hexamer and octamer has been proposed. Presumably, 50% of the active centers in the mitochondrial creatine kinase octamer are not involved in the catalytic act.     

Refined structure of southern bean mosaic virus at 2.9 A resolution

The T = 3 capsid of southern bean mosaic virus is analyzed in detail. The beta-sheets of the beta-barrel folding motif that form the subunits show a high degree of twist, generated by several beta-bulges. Only 34 water molecules were identified in association with the three quasi-equivalent subunits, most of them on the external viral surface. Subunit contacts related by quasi-3-fold axes are similar, are dominated by polar interactions and have almost identical calcium binding sites. There is no metal ion on the quasi-3-fold axis, as previously reported. Subunits related by quasi-2-fold and icosahedral 2-fold axes have different contacts but nevertheless display almost identical interactions between the antiparallel helices alpha A. A dipole-dipole type interaction between these helices may produce an energetically stable hinge that allows two types of dimers in a T = 3 assembly. The temperature factor distribution, the hydrogen-bonding pattern, and the contacts across the icosahedral 2-fold axes suggest that one of the dimer types is present in the intact virion and probably also in solution; the other is produced only during capsid assembly. Interactions along the 5-fold axes are mainly polar and possibly form an ion channel. The beta-sheet structures of the three subunits can be superimposed with considerable precision. Significant relative distortions between quasi-equivalent subunits occur mainly in helices and loops. The two dimeric forms and the subunit distortions are the consequence of the non-equivalent subunit environments in the capsid.     

Disulfide linkage of the b and delta subunits does not affect the function of the Escherichia coli ATP synthase

The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits. We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit. The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta. The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain. These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Lactose and D-galactose metabolism in Staphylococcus aureus. III. Purification and properties of D-tagatose-6-phosphate kinase

D-Tagatose-6-phosphate kinase, an inducible enzyme that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus, was purified about 300-fold from an extract of D-galactose-grown cells. The enzyme catalyzed the nucleoside triphosphate-dependent phosphorylation of both D-tagatose 6-phosphate and D-fructose 6-phosphate. Although the Vmax values were equal for these two substrates, the apparent Km values differed by 10,000-fold, being 16 micro M for D-tagatose 6-phosphate and 150 mM for D-fructose 6-phosphate. The purified enzyme was free from the constitutive D-fructose-6-phosphate kinase. Phosphoryl donors used by D-tagatose-6-phosphate kinse, listed in order of decreasing rates at saturating concentrations were GTP, UTP ITP ATP, CTP, and TTP; the Km values were 0.38, 0.91, 0.17, 0.16, 18, and 20 mM, respectively. The enzyme appeared to be nonallosteric; it exhibited Michaelis-Menten kinetics and was not inhibited by high concentrations of MgATP. However, it was activated 3- to 4-fold by 33.3 mM K+, NH4+, Rb+, and Cs+, and was inhibited 31 to 65% by 33.3 mM Na+ and Li+. It was inactivated reversibly by the thiol reagent, N-ethylmaleimide. The subunit molecular weight was estimated to be 52,000, and the native enzyme appeared to be a dimer with a sedimentation coefficient of 6.8 S. Data on stability, pH optimum, and inducibility of the enzyme are also presented.     

Free radical inactivation of glucose-6-phosphate dehydrogenase in Saccharomyces cerevisiae in vitro

Partial purification and in vitro inactivation of glucose-6-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae in the Fe2+/H2O2 oxidation system were conducted. At the protein concentration 1.5 mg/ml, the enzyme lost 50% of activity within 5 minutes of incubation in presence of 2 mM hydrogen peroxide and 3 mM ferrous sulphate. The inactivation extent depended on time and concentrations of FeSO4 and H2O2. EDTA, ADP and ATP at concentration 0.5 mM enhanced inactivation. At the same time, the presence of 0.5 mM NADPH, 1 mM glucose-6-phosphate, 10 mM mannitol, 30 mM dimethylsulphoxide or 20 mM urea diminished this process. In comparison with native enzyme, index S(0,5) of the partially inactivated enzyme for glucose-6-phosphate was 2.1-fold higher, but for NADP it was 1,6-fold lower. Maximal activity of the partially inactivated enzyme was 3-5-fold lower than that of native one.     

Electron microscopic analysis and biochemical characterization of a novel methanol dehydrogenase from the thermotolerant Bacillus sp. C1

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase.     

Crystal structure of the complex of phosphofructokinase from Escherichia coli with its reaction products

The crystal structure of Escherichia coli phosphofructokinase complexed with its reaction products fructose 1,6-bisphosphate (Fru1,6P) and ADP/Mg2+, and the allosteric activator ADP/Mg2+, has been determined at 2.4 A resolution. The structure was solved by molecular replacement using the known structure of Bacillus stearothermophilus phosphofructokinase, and has been refined to a crystallographic R-factor of 0.165 for all data. The crystallization mixture contained the substrate fructose 6-phosphate, but the electron density maps showed clearly the presence of the product fructose 1,6-bisphosphate, presumably formed by the enzyme reaction with contaminating ATP. The crystal consists of tetrameric molecules with subunits in two different conformations despite their chemical identity. The magnesium ion in the "closed" subunit bridges the phosphate groups of the two products. In the "open" subunit, the products are about 1.5 A further apart, with the Mg2+ bound only to ADP. These two conformations probably represent two successive stages along the reaction pathway, in which the closure of the subunit is required to bring the substrates sufficiently close to react. This conformational change within the subunit is distinct from the quaternary structure change seen previously in the inactive T-state conformation. It is probably not involved in the co-operativity or allosteric control of the enzyme, since the co-operative product fructose 1,6-bisphosphate is not moved, nor are the subunit interfaces changed. The structure of the enzyme is similar to that of B. stearothermophilus phosphofructokinase, and confirms the location of the sites for the two reaction products (or substrates), and of the effector site binding the activator ADP/Mg2+. However, this structure gives a clearer picture of the active site, and of the interactions between the enzyme and its reaction products.     

Purification and properties of myo-inositol-1-phosphatase from bovine brain.

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.     

Hemocyanin from Tachypleus gigas. II. Cooperative interactions of the subunits

Six subunits (I to VI) were isolated from hemocyanin of an Asian horseshoe crab, Tachypleus gigas, by anion exchange chromatography of the dissociated hemocyanin. The subunit preparations were nearly homogeneous as judged by alkaline electrophoresis, but they still showed the presence of isoproteins in isoelectric focusing. The subunits were reassembled (in 10 mM CaCl2 at pH 7.5) and tested for restoration of the cooperativity in O2 binding. The reassembly of the subunits gave equilibrium mixtures of the monomer and hexamer with small amounts of larger molecules. Homogeneous and heterogeneous hexamers were prepared by reassembling a single kind or two kinds of subunits, followed by isolation of the hexamer fraction by gel filtration. Among the homohexamers, only the subunit V hexamer showed cooperativity in O2 binding with the Hill coefficient of 1.6. Among the heterohexamers the subunit I/V hybrid was most noteworthy, showing a Hill coefficient (1.7) higher than that of any other heterohexamer examined. It was concluded that there are specific interactions between the subunits I and V. It is suggested that their interactions are important for the cooperativity in the native hemocyanin.