Biomedical subjects
Publications and source records attributed to E S Younathan.
Two polymorphs of the title compound, (4R,5R,6R,7R)-4,7-bis(hydroxymethyl)-1,3-dioxepane-5,6-diol, C(7)H(14)O(6), both have Z' = 2 at 100 K, and differ in their hydrogen-bonding patterns. The sodium iodide complex, NaI.C(7)H(14)O(6), is isomorphous with the NaCl complex, and has the mannitol, cation and anion all lying on twofold axes. The dioxepane rings of all three molecules are in the twist-chair conformation.
The central six-C-atom chain of the title compound, C52H50O10, adopts a nearly planar extended conformation free from C parallel C, C parallel O and O parallel O 1,3-parallel interactions. The three torsion angles formed by these atoms have values of 177.4 (2), 174.2 (2) and -178.0 (2) degrees. The bulky triphenylmethyl groups, which are oriented gauche to their neighboring acetoxy groups with O--C--C--O torsion angles of -69.8 (2) and -63.0 (2) degrees, cause no distortion of the bond lengths and bond angles of the sugar moiety.
The title compound, 2,5-bis(4-methylphenylsulfonyloxy-methyl)oxolane-3,4-diyl diacetate, C24H28O11S2, lies on a crystallographic twofold axis. It adopts a perfect twist 3(4)T conformation in the solid state. The puckering parameters of the tetrahydrofuran ring are q = 0.32 (8) A and rho = 92.3 (5) degrees. The acetyl groups are planar, with the non-H atoms deviating less than 0.002 (3) A from their mean plane. They have an (S)-cis conformation with the C-O and C = O bonds eclipsed, and each acetyl group is orientated with the C = O group syndiaxial to the C-H bond at the ring C atom to which the group is attached.
3,4-Di-O-acetyl-2,5-anhydro-1,6-dideoxy-1,6-diiodo-D-mannitol (3) is prepared from 2,5-anhydro-D-mannitol (1) in three steps. The solution and solid-state NMR spectra of 3 indicate considerable variation in conformation. In solution, it adopts, on average, a symmetric 4T3 conformation, whereas in the solid state it adopts an asymmetric conformation as revealed by 13C NMR cross polarization and magic angle spinning techniques. A single-crystal X-ray structure analysis confirmed the asymmetric conformation of 3 in a monoclinic crystal, space group P2(1) with a = 8.9608(4), b = 8.6348(5), c = 9.6468(4) A, beta = 96.139(4) degrees, V = 742.1(1) A3, Dc = 2.085 g cm-3, mu (MoK alpha) = 4.2 mm-1, and Z = 2. The structure was refined to R = 0.039 and Rw = 0.047 for 5181 observed reflections. The furanoid ring of 3 adopts an envelope E5 conformation slightly distorted towards 4T5, with puckering parameters psi = 313.49 degrees and q = 0.37 A. The asymmetric conformation is rationalized in terms of the weak packing forces in the crystal.
The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B. stearothermophilus PFK (BsPFK) is only slightly inhibited. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK. To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve. From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different. The chimeric subunit is locked in a structure resembling that of activated E. coli PFK, yet the enzyme can exist in two different conformational states. Mechanisms for its sigmoidal kinetics are discussed.
Complete analyses of the proton and carbon chemical-shift assignments of D-mannurono-gamma-lactone (1) have been achieved by 1D and 2D NMR spectral measurements. At equilibrium, the anomeric alpha and beta forms were present in the ratio of 34:66 in D2O and 72:28 in Me2SO-d6. The solution data indicated that the dienvelope conformation 2E:E4 to be the favored conformation of 1 in solution. The crystal structure of 1 was determined, and it showed that the crystalline form is the beta anomer, a bicyclic structure, consisting of fused five-membered furanose and lactone rings, in agreement with an earlier deduction from chemical evidence. In contrast to the solution conformation, the furanose ring adopts a twist conformation lying between the 2(1)T and 1E conformations with phase angle (P) and pseudorotation amplitude (tau m) of -44.23 degrees and 37.9 degrees, respectively, whereas the lactone ring adopts an envelope E5 conformation slightly distorted towards 6T5 with a phase angle (P) of -22.3 degrees and a pseudorotation amplitude (tau m) of 18.4 degrees. The molecules are linked in the crystal through a hydrogen-bonding network that involves all hydroxyl groups as well as the ring oxygen atoms.
The crystal structure of 3,4,3',4'-tetra-O-acetyl- 6,6'-di(triphenylmethyl)-di-D-fructose anhydride I (1) has been determined by single-crystal X-ray diffraction. The crystals are monoclinic, space group P2(1) with a = 16.399(2), b = 9.091(2), c = 17.946(4) A, beta = 103.66(1) degrees, V = 2600(2) A3, and Z = 2. The structure was refined to R = 0.044 and Rw = 0.051 for 4403 observed reflections. The structure analysis of 1 showed that the previously assigned chemical structure of di-D-fructose anhydride I is undoubtedly alpha-D-fructofuranose beta-D-fructofuranose 1,2':2,1'-dianhydride. The conformations of the furanose rings are E5 with P = 59.8 and tau m = 43.2 degrees for D-fructose 1, and 2T3 with P = -34.39 degrees and tau m = 39.64 degrees for D-fructose 2. The two furanose fragments are linked by a 1,4-dioxane ring in a spiro arrangement. The 1,4-dioxane ring has a chair conformation with Cremer-Pople puckering parameters Q = 0.527 A, phi = 72.2 degrees and the a = 14.2 degrees.
Methyl 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranoside, C13H20O8, M(r) = 304.3, is monoclinic, space group C2, with a = 23.619(1), b = 8.2168(5), c = 19.093(1) A, beta = 118.72(1) degrees, V = 3249.6(8) A3, Dc = 1.244 g cm-3, mu (MoK alpha) = 0.97 cm-1 and Z = 8. The structure was refined to R = 0.044 and Rw = 0.039 for 1969 observed reflections. There are two independent molecules in the asymmetric unit. The bond lengths and bond angles of the pyranose rings of the two are in good agreement within the limits of error. The molecules have similar conformation except for the orientation of one of the acetoxy groups. Each molecule is a normal 1C4 chair with Cremer-Pople puckering parameters Q = 0.557(6) A, theta = 174.6(2) degrees and psi = 144.6(9) degrees for molecule A and 0.564(4) A, 177.9(1) degree and 30.8(8) degrees for molecule B, respectively. The acetyl groups have the planar, (S)-cis conformation most commonly observed. They are oriented with the acetyl planes within +/- 35 degrees of the C-H bond at the ring carbon atom to which they are attached.
The crystal structures of L-rhamnono-1,4-lactone (1) and L-mannono-1,4-lactone (2) have been determined by single-crystal X-ray diffraction. Pertinent crystal data are as follows: for 1, orthorhombic, space group P2(1)2(1)2(1), a = 4.8829(2), b = 10.9088(8), c = 13.9758(9) A, V = 734.7(1) A3, Dc = 1.610 g cm-3, Z = 4, R = 0.028 and Rw = 0.035 for 1586 reflections. The lactone ring of 1 adopts an envelope conformation, E3, slightly distorted toward 2T3, with psi = 103.1(7) degrees and q = 0.38(3) A, whereas the lactone ring of 2 adopts a perfect envelope E3 conformation, with psi = 106.6(4) degrees and q = 0.42(4) A. Molecules of 1 and 2 are linked in their crystals through a three dimensional network of O-H ... H hydrogen-bonding interactions that involves all hydroxyl groups as well as the carbonyl oxygen atom.
The crystal structure of 2-C-methyl-D-ribo-pentono-1,4-lactone (alpha-D-glucosaccharino-gamma-lactone, 1) has been determined by single-crystal X-ray diffraction. The crystals are orthorhombic, space group P2(1)2(1)2(1) with a = 7.7429(6), b = 8.3373(7), c = 11.3258(7) A, V = 731.1(2) A3 (CuK alpha, lambda = 1.54184 A), mu = 10.82 cm-1, Dc = 1.473 g cm-3, and Z = 4. The structure was refined to R = 0.0307 and Rw = 0.0424 for 876 observed reflections. Compound 1 has the D-ribo configuration, in agreement with an earlier deduction from chemical evidence. The lactone ring adopts the 3T2 conformation, with puckering parameters psi = 279.8(9) degrees and q = 0.32(5) A. The orientation of the methyl group about the C-2-C-3 bond is gauche-trans, with the C-6-C-2-C-3-O-3 and C-6-C-2-C-3-C-4 torsion angles being -81.3(2) degrees and 154.7(1) degree, respectively. The molecules are linked in the crystal in a two-dimensional intermolecular hydrogen bonding network that involves all hydroxyl groups as well as the carbonyl oxygen atom.
1,2-O-isopropylidene-alpha-D-xylo-pentodialdo-1,4-furanose (1), C16H24O10, M(r) = 376.4, is orthorhombic, space group P2(1)2(1)2(1) with a = 10.3028(10), b = 11.1875(3), c = 15.7484(13) A, V = 1815.2(4) A3, Dc = 1.377 gcm-3, mu(CuK alpha) = 9.5 cm-1 and Z = 4. The structure was refined to R = 0.033 and Rw = 0.045 for 1984 observed reflections. Crystalline 1 has a dimeric cyclic acetal-hemiacetal structure, formed by self aldol condensation of two monomers. The absolute configuration at the condensation centers, C-5 and C-5', were assigned as 5R and 5S, respectively. In the dimer 1, the xylofuranose rings adopt different conformations, one is a twist 3T4, whereas the second is an envelope E4 slightly distorted towards the 3T4 conformation; their fused 1,2-O-isopropylidene rings adopt O-2E and O-2TC-6 conformations, respectively. The 1,3-dioxane ring has a distorted chair conformation with puckering parameters Q = 0.516 A, phi = 90.9, and theta = 11.0 degrees. The molecules are linked in the crystal through intermolecular hydrogen-bonding interactions that involve the two hydroxyl groups, OH-3 and OH-5', and the isopropylidene ring oxygen atoms, O-2 and O-1', as donor and acceptor, respectively.
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The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.
2,5-Anhydro-1-O-(p-tolylsulfonyl)-D-mannitol, C13H18SO7, Mr = 318.4, monoclinic, C2, a = 26.370(6), b = 7.9741(11), c = 6.6801(6) A, beta = 91.401(11) degrees, V = 1404.3(6) A3, Z = 4, DX = 1.506 g/cm3, CuK alpha, lambda = 1.54184 A, mu = 23.03 cm-1, F(000) = 672, T = 296(1) K, R = 0.042 for 2832 observations with I > 3 sigma (I) (of 2864 unique data). On the esterified side of the molecule, three bond lengths and three bond angles show small changes compared to the unesterified side, which is similar to the symmetrical parent compound, 2,5-anhydro-D-mannitol. The conformation of the five-membered ring is E5 with P = 49.3 degrees and tau m = 38.1 degrees. The hydroxymethyl groups adopt g+ and g- dispositions similar to the parent molecule. The three hydroxyl groups are involved in a network of intermolecular hydrogen bounds both as donors and acceptors.
The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.
3,6-Dioxabicyclo[3.1.0]hexane-2,4-dimethanol, C6H10O4, M(r) = 146.1, orthorhombic, P2(1)2(1)2(1), a = 7.6209 (2), b = 9.1292 (3), c = 9.6135 (5) A, V = 668.8 (1) A3, Z = 4, Dx = 1.451 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 1.15 cm-1, F(000) = 312, T = 298 K, R = 0.029 for 1280 observations with I greater than 3 sigma(I) (of 1695 unique data). The tetrahydrofuran ring has the envelope conformation, OE, with P of 94.3 degrees and tau m = 24.0 degrees. C atoms deviate from their best plane by +/- 0.0006 (1) to 0.010 (1) A, and the O atom lies 0.331 (1) A from that plane. The epoxide O atom is syn to the tetrahydrofuran O atom. Each hydroxy group is involved in intermolecular hydrogen bonding both as donor and acceptor. The two hydrogen bonds have O...O distances of 2.743 (1) and 2.729 (1) A, and angles about H of 166.3 (12) and 172 (2) degrees, respectively.
2,5-Anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol (1) was prepared from 2,5-anhydro-D-mannitol (2) in three steps. The fused ring system was introduced by a phase-transfer alkylation using 1,2-dibromoethane. Its conformation in solution was determined by NMR studies at 500 MHz. Variable-temperature studies showed no lineshape change from 25 to 80 degrees in D2O. The data indicate that the five-membered ring is locked by the trans-fused six-membered 1,4-dioxane ring into a twist 4T3 conformation. A single-crystal X-ray study was carried out. The crystals are orthorhombic, C222(1), a = 4.7252 (6), b = 14.0364 (12), c = 13.268 (2) A, Z = 4, with R = 0.032 for 894 observations. The molecule lies upon a crystallographic two-fold axis, and thus the five-membered ring exists in a perfect 4T3 conformation with a pseudorotation angle of 0 degree and amplitude of 47.2 degrees, in agreement with the NMR results. We have shown earlier that, among twenty possible conformers, phosphofructokinase acts specifically on the 4T3 conformer of the beta anomer of D-fructose 6-phosphate.
C18H24O12, Mr = 432.4, monoclinic, Cc, a = 8.996 (3), b = 20.890 (6), c = 11.872 (4) A, beta = 101.11 (2) degrees, V = 2189 (1) A3, Z = 4, Dx = 1.312 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 1.05 cm-1, F(000) = 912, T = 163 K, R = 0.041, wR = 0.0375 for 2158 reflections (Fo greater than or equal to 6 sigma magnitude of Fo). The ring is in the chair conformation 4C1 with five equatorial groups and one axial group bonded to C(2) as expected. The carbonyl bonds of the acetate groups at positions 2, 4, 5 and 6 are approximately coplanar with their respective ring C-H bonds. However, those at positions 1 and 3 are rotated towards the H(2) atom.