Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.     

1H-NMR studies on nucleotide binding to the sarcoplasmic reticulum Ca2+ ATPase. Determination of the conformations of bound nucleotides by the measurement of proton-proton transferred nuclear Overhauser enhancements

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.     

Crystal structures of aspartate carbamoyltransferase ligated with phosphonoacetamide, malonate, and CTP or ATP at 2.8-A resolution and neutral pH

The R-state structures of the ATP and CTP complexes of aspartate carbamoyltransferase ligated with phosphonoacetamide and malonate have been determined at 2.8-A resolution and neutral pH. These structures were solved by the method of molecular replacement and were refined to crystallographic residuals between 0.167 and 0.182. The triphosphate, the ribose, and the purine and pyrimidine moieties of ATP and CTP interact with similar regions of the allosteric domain of the regulatory dimer. ATP and CTP relatively increase and decrease the size of the allosteric site in the vicinity of the base, respectively. For both CTP and ATP at pH 7, the gamma-phosphates are bound to His20 and are also near Lys94, while the alpha-phosphates interact exclusively with Lys94. The 2'-hydroxyls of both CTP and ATP are near the amino group of Lys60. The pyrimidine ring of CTP makes specific hydrogen bonds at the allosteric site: the NH2 group donates hydrogen bonds to the main-chain carbonyls of Ile12 and Tyr89 and the pyrimidine ring carbonyl oxygen accepts a hydrogen bond from the amino group of Lys60; the nitrogen at position 3 in the pyrimidine ring is hydrogen bonded to a main-chain NH group of Ile12. The purine ring of ATP also makes numerous interactions with residues at the allosteric site: the purine NH2 (analogous to the amino group of CTP) donates a hydrogen bond to the main-chain carbonyl oxygen of Ile12, the N3 nitrogen interacts with the amino group of Lys60, and the N1 nitrogen hydrogen bonds to the NH group of Ile12. The binding of CTP and ATP to the allosteric site in the presence of phosphonoacetamide and malonate does not dramatically alter the structure of the allosteric binding site or of the allosteric domain. Nonetheless, in the CTP-ligated structure, the average separation between the catalytic trimers decreases by approximately 0.5 A, indicating a small shift of the quaternary structure toward the T state. In the CTP- and ATP-ligated R-state structures, the binding and occupancy of phosphonoacetamide and malonate are similar and the structures of the active sites are similar at the current resolution of 2.8 A.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Structure of human oxyhaemoglobin at 2.1 A resolution

The structure of human oxyhaemoglobin was determined by single crystal X-ray analysis at 2.1 A resolution. Data were collected on an Arndt-Wonacott camera at -2 degrees C. The structure was refined to an R factor of 0.223 by the Jack-Levitt method, starting from Baldwin's model of human carbon monoxide haemoglobin. The active sites in the alpha and beta subunit are distinct. The iron atoms are 0.16(8) A and 0.00(8) A from the mean plane of the porphyrin carbons and nitrogens (0.12(8) A and -0.11(8) A from the mean plane of the porphyrin nitrogens) in the alpha and beta subunit, respectively, in correlation with the orientation of HisF8 relative to the porphyrin nitrogens. The haem group appears to be nearly planar in the alpha subunit but ruffled in the beta subunit. The Fe-O(1)-O(2) angles are 153(7) degrees and 159(12) degrees in the alpha and beta subunit, respectively. The oxygen molecule forms a hydrogen bond to N epsilon of HisE7 in the alpha, but either none or a weak one in the beta subunit. The following bond lengths were found: Fe-N epsilon (HisF8) = 1.94(9) A (alpha) and 2.07(9) A (beta); Fe-O(1) = 1.66(8) A (alpha) and 1.87(13) A (beta); Fe-Nporph (mean = 1.99(5) A (alpha) and 1.96(6) A (beta). These dimensions agree with the values obtained in oxymyoglobin and model compounds. The C-terminal residues, ArgHC3(141 alpha) and HisHC3(146 beta), are relatively delocalized, and their positions do not enable them to form the intersubunit salt bridges in which they are involved in deoxyhaemoglobin. The penultimate tyrosine residues, TyrHC2 140 alpha and 145 beta, are relatively localized and maintain the hydrogen bonds to the carbonyl oxygens of ValFG5 (93 alpha and 98 beta), with only minor variations compared to their geometry in deoxyhaemoglobin. TyrHC2(145 beta), however, alternates between a major and a minor site, in conjunction with CysF9(93 beta), both sharing the internal pocket between the F and H helices while in the major conformation. This suggests that the role of the penultimate tyrosines in the allosteric mechanism may differ from that previously proposed by Perutz. The overall quaternary structure of oxyhaemoglobin is identical, within experimental error, to that of carbon monoxide haemoglobin, and thus confirms the applicability of the allosteric mechanisms proposed by Perutz and Baldwin & Chothia to the process of oxygen binding.     

Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)beta leads to Ser).

A difference Fourier synthesis of deoxyhaemoglobin Tacoma minus deoxyhaemoglobin A at 3.5 Å resolution has been calculated. The map shows a large negative peak due to the removal of the guanidinium group of Arg B12(30)β, surrounded by positive and negative peaks indicative of some atoms moving towards, and others away from, the vacated site. Among the latter, the most important is the carboxylate of Glu B8(26)β which is hydrogen-bonded to the guanidinium group of the arginine in haemoglobin A, but swings round its α-β carbon bond towards the imidazoles of histidines G18(116) and 19(117)β in haemoglobin Tacoma. This movement would raise the pK values of the histidines, so that their positive charges compensated for the loss of the arginine. This may explain why haemoglobin Tacoma has the same electrophoretic mobility as haemoglobin A. It is shown that haemoglobin Tacoma has a lower oxygen equilibrium constant K_T and a larger allosteric constant L than haemoglobin A. The lowering of K_T may be due to the loosening of the T structure in haemoglobin Tacoma consequent upon the removal of the hydrogen bonds made by the guanidinium group of Arg B12(30)β at the α₁β₁ contact. Their removal also accounts for the decreased stability of haemoglobin Tacoma. We cannot yet explain its diminished Bohr effect, nor the increase in L.     

Influence of hyperosmotic shrinkage and β-adrenergic stimulation on red blood cell volume regulation and oxygen binding properties in rainbow trout and carp

Whole blood from rainbow trout and carp was subjected to hyperosmotic shock and subsequent beta-adrenergic stimulation (isoprenaline) at different oxygen tension ( PO(2)) and carbon dioxide tension ( PCO(2)) levels with the aim to evaluate changes in red blood cell (RBC) volume, pH and ion concentrations and their ultimate effect on blood O(2) transport characteristics. Hyperosmolality (addition of NaCl) induced RBC shrinkage, which was followed by a regulatory volume increase (RVI) that was larger at low than at high PO(2)and more complete in carp than in trout. Carp RBC showed practically full volume recovery within 140 min at low PO(2)and partial recovery at high PO(2), whereas RVI was partial under all PO(2)and PCO(2)conditions in trout. The RVI increased intracellular [Na(+)], water content, and, in carp, also pH (pHi), suggesting activation of Na(+)/H(+) exchange. In trout RBCs, activation of RVI was rapid but succeeded by deactivation. In carp RBCs, activation of Na(+) influx was slower but it continued, allowing full volume recovery. Shrinkage of the RBCs was associated with only minor decreases in blood oxygen saturation and oxygen affinity in both species. Thus, the oxygen affinity decrease expected on the basis of the increased concentration of intracellular haemoglobin and organic phosphates was small, and it appeared to some extent countered during RVI by an oxygen affinity increase via increased pHi. Addition of isoprenaline increased RBC volume and pHi and increased Hb oxygen saturation. The beta-adrenergic response was stronger at low compared to high PO(2) and at high compared to low PCO(2). The PO(2) dependency was largest in carp, whereas the PCO(2) (pH) dependency was more expressed in trout. The adrenergic response of trout RBCs was similar under isoosmotic and hyperosmotic conditions. In carp RBCs, the response was absent at high PO(2) under isoosmotic conditions, but interestingly it could be induced under hyperosmotic conditions. The data suggest that the RBC shrinkage occurring in fish moving from freshwater to seawater has minimal impact on blood O(2) binding properties.     

The effects of various GTP analogues on microtubule assembly.

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.     

The Influence of Salinity Acclimation on Free Amino Acids and Enzyme Activities in the Intestinal Mucosa of Rainbow Trout,Oncorhynchus mykiss (Walbaum)

Postprandial changes of Arg, Leu, Val, Ala, Asp, Glu, Gly, Pro and Tau as well as activities of three enzymes of the transdeamination system in the midgut mucosa and, for comparison, in the liver of freshwater and seawater acclimated Oncorhynchus mykiss were studied. In the mucosa a postprandial increase of Arg, Leu, Val, Ala, Asp, Glu, Gly and Pro occurred. In contrast, only the postprandial Arg level increased strongly in the liver. Levels of Leu, Val, Ala, Asp, Glu, Gly, Pro and Tau remained stable. Concentrations of Ala, Asp, Glu and Pro are higher in the liver than the mucosa. Tau is the most important osmotic effector in both organs, but its concentration is much lower in the liver. Its postprandial concentrations remained stable in both tissues but were significantly higher in seawater trout. The trend of a stronger postprandial rise of Arg, Leu, Val, Ala, Asp, Glu, Gly and Pro levels in seawater trout than in freshwater trout was shown. In mucosa tissue aspartate aminotransferase activities were higher in seawater trout. Ratios of aspartate aminotransferase, alanine aminotransferase and glutamate dehydrogenase are similar to those of the gills.     

AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.     

Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Identification of the geometric requirements for allosteric communication between the alpha- and beta-subunits of tryptophan synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.     

Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg).

Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.     

The structure and antiviral activity of ribavirin analogs. II. Molecular and crystal structure of 1-(1,5-dihydroxy-3-oxapent-2-yl)-1,2,4-triazole-5-carboxamide

X-ray structure of the title compound, an antiviral agent moderately active towards Herpes simplex virus type 1, has been determined. The space group is P2i/n, unit cell parameters: a = 10,119, b = 7,529, c = 13,585 A, beta = 107,82 degrees, Z = 4. The structure was solved by the direct method and refined by least-squares procedure to R = 2.9%. The gauche-conformation about C4'-C5' bond and trans-conformation about O4'-C4' bond are realized in the molecule. The carboxyamide group at the C5 atom of triazol cycle provides a steric opportunity for the intramolecular hydrogen bond C1'-H1'...O6 formation.     

Characterization of the interactions of NADH with the dimeric and tetrameric states of methaemoglobin

The binding of NADH to the dimeric (αβ) and tetrameric (α₂β₂) states of human aquomethaemoglobin has been characterized by sedimentation equilibrium studies of the effect of the concentration of free ligand on the macromolecular state of the haemoprotein. Both macromolecular states of aquomethaemoglobin exhibit a single binding site for NADH, which interacts approximately tenfold more strongly (6000 cf. 700 M⁻¹) with the tetramer under the conditions studied (pH 6.0, I 0.10). Because the structure of aquomethaemoglobin resembles that of the deoxy state of haemoglobin, there is a high probability that organic phosphates also bind to dimeric deoxyhaemoglobin, a phenomenon which is not considered in thermodynamic treatments of the interplay between oxygen binding and its allosteric inhibition by 2,3-bisphosphoglycerate. Fortunately, the equilibrium constant for deoxyhaemoglobin self-association is so large that neglect of the interaction between allosteric inhibitor and dimeric haemoglobin is an oversight that should have no deleterious implications in the resultant thermodynamic analysis of the interplay between the preferential interactions of oxygen and organic phosphate with the various macromolecular states of deoxyhaemoglobin.     

The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates

Most vertebrates die within minutes when deprived of molecular oxygen (anoxia), in part because of cardiac failure, which can be traced to an inadequate matching of cardiac ATP supply to ATP demand. Cardiac power output (PO; estimated from the product of cardiac output and central arterial pressure and an indirect measure of cardiac ATP demand) is directly related to cardiac ATP supply up to some maximal level during both normoxia (ATP supply estimated from myocardial O(2) consumption) and anoxia (ATP supply estimated from lactate production rates). Thus, steady state PO provides an excellent means to examine anoxia tolerance strategies among ectothermic vertebrates by indicating a matching of cardiac glycolytic ATP supply and demand. Here, we summarize in vitro measurements of PO data from rainbow trout, freshwater turtles and hagfishes to provide a reasonable benchmark PO of 0.7 mW g(-1) for maximum glycolytic potential of ectothermic hearts at 15 degrees C, which corresponds to a glycolytic ATP turnover rate of about 70 nmol ATP g(-1) s(-1). Using this benchmark to evaluate in vivo PO data for hagfishes, carps and turtles, we identify two cardiac survival strategies, which in conjunction with creative waste management techniques to reduce waste accumulation, allow for long-term cardiac survival during anoxia in these anoxia-tolerant species. Hagfish and crucian carp exemplify a strategy of evolving such a low routine PO that routine cardiac ATP demand lies within the range of the maximum cardiac glycolytic potential. Common carp and freshwater turtles exemplify an active strategy of temporarily and substantially decreasing cardiac and whole body metabolism so that PO is below maximum cardiac glycolytic potential during chronic anoxia despite being quite close to this potential under normoxia.     

The structure and antiviral activity of ribavirin analogs. I. Molecular and crystal structure of 1-(2-hydroxyethoxymethyl)-1,2,4-triazole-5-carboxamide

The crystal and molecular structures of the antiviral compound 1-(2-hydroxyethoxymethyl)-1,2,4-triazole-5-carboxamide has been determined by the X-ray diffraction method. The space group is P2i/c, unit cell parameters a = 4,381, b = 18,679, c = 10,776 A, beta = 107,40 degrees, Z = 4. The structure was solved by the direct method and refined by a full-matrix least-squares procedure to R = 4.9%. Two planar groups of atoms can be distinguished in the molecule. The first group involves the atoms of triazole ring, C6, and C1', the second one contains C5, C6, O6 and N6 atoms. The angle between these planes is 5.6 degrees. The carboxyamide group is rotated by 180 degrees in comparison with this group in ribavirin. That is why the intramolecular hydrogen bond C1'-H1'. 1...O6 can form. Torsion angle O5'-C5'-C4'-O4' is 73.9 degrees and it corresponds to gauche-rotamer. The conformation about O4'-C4' bond is trans. The C1'-C4' bond is approximately perpendicular to the aglycone.     

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.     

Interactions of adenosine triphosphate with snake hemoglobins. Studies in Liophis miliaris,Boa constrictor and Bothrops alternatus

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about K_D ≅ 10⁶ M⁻¹ (10⁷ M⁻¹ for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about K_O ≅ 10² M⁻¹. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about K_D ≅ 10³ M⁻¹ whereas for the oxyhemoglobin the constants remain of about the same, K_O ≅ 10² M⁻¹, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.