Allosteric Function Research Articles

Substitution of Ser for Arg-443 in the Regulatory Domain of Human Housekeeping (GLUD1) Glutamate Dehydrogenase Virtually Abolishes Basal Activity and Markedly Alters the Activation of the Enzyme by ADP and l-Leucine

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by l-leucine (1-10 mm) in the absence of other effectors. However, when ADP (0.025-0.1 mm) was present in the reaction mixture, l-leucine (0.3-6.0 mm) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.

S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 +/- 17 nm in human arterial erythrocytes and 69 +/- 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the beta-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.

Molecular Mechanism for the Regulation of Human Mitochondrial NAD(P) +-Dependent Malic Enzyme by ATP and Fumarate

The regulation of human mitochondrial NAD(P) +-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 Å resolution of m-NAD-ME in complex with ATP, Mn 2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.

Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium.

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.

Atypical binding of the neuronal POU protein N-Oct3 to noncanonical DNA targets. Implications for heterodimerization with HNF-3 beta.

The capacity of POU proteins to recognize different DNA sequences and to bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATGC) within the (-102/-72) neuronal promoter region of the human aromatic L-amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N-Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription activator HNF-3 beta. CD studies indicate that the binding of N-Oct3 monomers/dimers and N-Oct3-HNF-3 beta heterodimers to the DNA induces conformational changes of both protein and DNA. Partial proteolysis/MALDI-MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N-Oct3/HNF-3 beta/DNA ternary complex predicts a nucleotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site-dependent and site-independent protein and DNA conformational changes, general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.

On the role of the N-terminal group in the allosteric function of glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosphate. It is a homohexamer and has six allosteric sites located in clefts between the subunits. The amino acid side-chains in the allosteric site involved in phosphate binding are Arg158, Lys160 and Ser151 from one subunit and the N-terminal amino group from the facing polypeptide chain. To study the functional role of the terminal amino group, we utilized a specific non-enzymic transamination reaction, and we further reduced the product with borohydride, to obtain the corresponding enzyme with a terminal hydroxy group. Several experimental controls were performed to assess the procedure, including reconditioning of the enzyme samples by refolding chromatography. Allosteric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein. Its kinetic study suggests that the allosteric equilibrium for this modified enzyme is displaced to the R state, with the consequent loss of co-operativity. The deaminase with a terminal hydroxy acid, obtained by reducing the transaminated enzyme, showed significant recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme. It had lost, however, the pH-dependence of homotropic co-operativity shown by the unmodified deaminase in the pH range 6–8. These results show that the terminal amino group plays a part in the co-operativity of the enzyme and, more importantly, indicate that the loss of this co-operativity at low pH is due to the hydronation of this amino group.

High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70

The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383–540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 Å for the backbone atoms and 0.76 Å for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions.

Mutations in the substrate binding domain of the Escherichia coli 70 kda molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling

In Escherichia coli, DnaK is essential for the replication of bacteriophage λ DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405–468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate bacteriophage λ in the background of a dnaK deficient cell line, BB1553. This initial screen identified several proteins which were mutant at multiple positions. The multiple mutants were further dissected into single mutants which remained negative for λ propagation. Four of these single-site mutants were purified and assayed for biochemical functionality. Two single-site mutations, F426S and S427P, are localized in the peptide binding site and display weakened peptide binding affinity. This indicates that the crystallographically determined peptide binding site is also critical for in vivo λ replication. Two other mutations, K414I and N451K, are located at the edge of the β-sandwich domain near α-helix A. The K414I mutant binds peptide moderately well, yet displays defects in allosteric functions, including peptide-stimulated ATPase activity, ATP-induced changes in tryptophan fluorescence, ATP-induced peptide release, and elevated ATPase activity. The K414 position is close in tertiary structure to the linker region to the ATPase domain and reflects a specific area of the peptide-binding domain which is necessary for interdomain coupling. The mutant N451K displays defects in both peptide binding and allosteric interaction.

Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase.

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments. Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme [Altamirano et al. (1994) Eur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket [Oliva et al. (1995) Structure 3, 1323-1332]. Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects. Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosamine 6-P deaminase.

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.

Regulation of GABAA Receptor Structure and Function by Chronic Drug Treatments In Vivo and with Stably Transfected Cells

In this article, we review the use of stably transfected cells to study the regulation of receptor structure and function by chronic drug treatments and compare results from these cells to results obtained from other systems, including neuronal cultures and intact animals. We focus on the gamma-aminobutyric acid type A (GABAA) receptor complex. Sedative/hypnotic drugs such as benzodiazepines, barbiturates and alcohol that potentiate GABAA receptor function produce tolerance and dependence. Chronic treatment of GABAA receptor preparations from brain and neuronal cultures with GABAA agonists, as well as these other three classes of drugs, results in regulation of several properties of the receptor. Drug treatments may regulate levels of binding sites, allosteric binding interactions, receptor function, levels of receptor subunit mRNA and levels of receptor subunit protein. Some or all of these effects may comprise the molecular mechanisms of tolerance to these GABAA-modulatory drugs. The use of cells stably transfected with neurotransmitter receptors provides a homogeneous population that can be cultured under controlled conditions. As most preparations contain mixed populations of GABAA receptor subunits, stably transfected cells offer the advantage of the expression of receptors with a defined subunit composition. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells. This regulation does not appear to be due to decreases in the expression of alpha 1- or beta 1-receptor subunits or to expression of subunits other than alpha 1, beta 1, gamma 2L. Therefore, it is unlikely to be due to changes in receptor subunit composition and probably represents post-translational changes. The rapid regulation of allosteric coupling and function by drug treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as tolerance and dependence of benzodiazepines and ethanol.

Asymmetric allosteric activation of Escherichia coli glucosamine-6-phosphate deaminase produced by replacements of Tyr 121

Tyrosine 121, a residue located in a alpha-helical polypeptide segment of glucosamine 6-phosphate deaminase from Escherichia coli, has recently been proposed to have a role in the binding of the allosteric activator N-acetyl-D-glucosamine 6-phosphate. Accordingly, the site-directed mutants Tyr 121-Thr and Tyr 121-Trp were constructed, to assess experimentally the role of Tyr 121 in the allosteric function of the enzyme. The kinetic study of both mutant forms revealed that the replacements caused striking changes in allosteric activator binding and allosteric properties, when compared to the wild-type enzyme. While the wild-type deaminase behaves as a classical allosteric K-system which can be described by the allosteric concerted model, both mutant forms present an asymmetric behavior toward the allosteric activator, which can be described as two distinct half-of-the-sites allosteric activation steps occurring with different affinities for the N-acetyl-D-glucosamine 6-phosphate. During the first (high affinity) activation phase, the mutant forms of deaminase behave as mixed K/V allosteric enzyme. The biphasic activation curve was also demonstrated by direct binding measurements of the 14C-labeled activator to Tyr 121-Trp and Tyr 121-Thr deaminases. The kinetic analysis of these mutant forms also showed that the threonine replacement produced an important distortion of the enzyme structure reflected in a considerable decrease of its catalytic efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of allosteric binding and function of stably expressed GABAA receptors by chronic treatment with ethanol. GABAA and benzodiazcpine agonists

Department of Pharmacology, University of Colorado Health Science Center, Denver, CO 80262, USA; 1Merck Sharp and Dohme Research Laboratories, Harlow, Essex, CM20 2PT, UK.

Oxygen equilibrium studies of cross-linked iron-cobalt hybrid hemoglobins. Models for partially ligated intermediates of cobalt hemoglobin.

To probe the molecular mechanism of allosteric function of cobaltous protoporphyrin-substituted hemoglobin (CoHb), a series of alpha, alpha-cross-linked symmetric and asymmetric Fe-Co hybrid hemoglobins, which contain (1Co porphyrin/3Fe porphyrins), (2Co porphyrins/2Fe porphyrins), and (3Co porphyrins/1Fe porphyrin) per tetramer, have been prepared. Because only Fe porphyrin-containing subunits react with CO, these Fe-Co hybrids are converted to mono-, di-, and tri-CO-ligated states in the presence of CO, respectively, and are proposed to stand as models for mono-, di-, and tri-ligated intermediates of CoHb, respectively. The oxygen binding properties of these Fe-Co hybrids were investigated by measuring oxygen binding isotherms in the presence of CO as a function of pH in the presence and absence of IHP. The ligation of CO to a beta subunit causes larger changes in the oxygen affinity and the Bohr effect than that to an alpha subunit, indicating that the ligation to a beta subunit induces larger affinity-related structural changes in cross-linked CoHb. Di- and tri-CO-ligated intermediates exhibited substantially increased oxygen affinity, reduced Bohr effect, and reduced IHP effect, indicating that they are in high affinity states. Calculation of the Adair equilibrium constants for the first and last oxygenation steps for each of these intermediates permitted the determination of the level of free energy of cooperation. The intermediately ligated species of cross-linked CoHb are distributed in multiple levels of free energy of cooperation within the free energy difference of 1.14 kcal mol-1 between deoxy and fully ligated states of cross-linked CoHb at pH 7.4. The ligation process in CoHb is determined by the number and distribution of the bound ligands, and ligation takes place through steps that require minimal free energy changes.

Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea.

Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid C109 to histidine and a change of E119 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tri-partate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+UTP. E119D was inhibited by both CTP and CTP+UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. E119D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the S values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isoelectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantially from those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; E119D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.

Crystal structure of hexameric haemocyanin from Panulirus interruptus refined at 3.2 Å resolution

The use of non-crystallographic symmetry restraints in the refinement of the haemocyanin hexamer from Panulirus interruptus at 3.2 Å resolution has resulted in a final model with a very reasonable geometry and a crystallographic R-factor of 20.1 %, using 59,193 observed structure factor amplitudes between 8.0 and 3.2 Å. The mean co-ordinate error is approximately 0.35 Å. The six subunits appear to be related by symmetry operations that differ slightly from 32 point group symmetry. The six subunits have essentially maintained the same structure. The hexamer, with point group 32, is best described as a trimer of “tight dimers”. The contacts between the subunits in such a dimer are more numerous, and better conserved during evolution than contacts in a trimer. The interface of a tight dimer is separated by an internal cavity into two “contact areas”. The contact area nearest to the centre of the hexamer is most extensive and consists mainly of residues that are quite conserved among arthropodan haemocyanins. All these residues are provided by the second domain of each subunit. Hence, this second domain may play a crucial role in the allosteric functioning of this oxygen transport protein. The dinuclear copper oxygen-binding site resides in the centre of domain 2. This oxygen-binding centre is not fully accessible from the solvent. Three large cavities occur, however, within each subunit at the interfaces of the three domains. All three cavities contain ordered water molecules, and two of them are accessible from the surrounding solvent. These cavities may play a role in facilitating fast movement of dioxygen towards the binding site, which is situated in a highly conserved, rather hydrophobic core. A detailed definition of the geometry of the copper site is, of course, not possible at the limited resolution of 3.2 Å. Nevertheless, it is possible to conclude that each copper is co-ordinated by two, more or less tightly bound, histidine ligands and one more distant histidine residue. The six histidine residues utilize their N ε atoms for copper co-ordination, while their N δ atoms are engaged in hydrogen bonds with conserved residues or water molecules. The two distant histidine ligands are located in apical positions and are on opposite sides with respect to the plane approximately defined by the four more tightly bound histidine ligands and the two copper ions. The copper-to-copper distance is 3.5 to 3.6 Å in four of the subunits, but this distance deviates considerably in two others. The presence or absence of a small exogenous copper-binding ligand in this plane, such as a hydroxyl ion, is discussed.

Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product.

In Saccharomyces cerevisiae the anaerobic (oxygen-repressed) ANB1 gene and a group of aerobic (oxygen-induced) genes are coordinately regulated by the ROX1 gene. We report here that heme, known as an inducer of aerobic genes, also causes inhibition of ANB1 expression. Thus, in combination with the ROX1 gene product heme has an opposite effect on the expression of anaerobic and aerobic genes. Accumulation of ANB1 mRNA was sharply decreased in anaerobic cells grown in the presence of heme. This effect must operate at the level of transcription since heme also inhibited accumulation of CYC1 mRNA from an ANB1-CYC1 fusion. Heme precursors did not appear to function either as inhibitors or as activators. Oxygen itself also had no effect on transcription of ANB1. Repression by heme cannot be attributed to the respiratory competence conferred by heme since both ANB1 and the aerobic genes tr-1 and CYC1 were regulated normally in [rho 0] mutants. The results are consistent with a classical allosteric coeffector function for heme, although more indirect explanations are tenable. A role for the ROX1 gene product in transcriptional regulation can be inferred from the observation that there was no inhibition of ANB1 expression by heme in rox1 mutants. Judging from this epistasis the rox1 phenotype is not due to a defect in heme production; this would indicate that the ROX1 factor functions by mediating the effect of heme on transcription.

Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product.

In Saccharomyces cerevisiae the anaerobic (oxygen-repressed) ANB1 gene and a group of aerobic (oxygen-induced) genes are coordinately regulated by the ROX1 gene. We report here that heme, known as an inducer of aerobic genes, also causes inhibition of ANB1 expression. Thus, in combination with the ROX1 gene product heme has an opposite effect on the expression of anaerobic and aerobic genes. Accumulation of ANB1 mRNA was sharply decreased in anaerobic cells grown in the presence of heme. This effect must operate at the level of transcription since heme also inhibited accumulation of CYC1 mRNA from an ANB1-CYC1 fusion. Heme precursors did not appear to function either as inhibitors or as activators. Oxygen itself also had no effect on transcription of ANB1. Repression by heme cannot be attributed to the respiratory competence conferred by heme since both ANB1 and the aerobic genes tr-1 and CYC1 were regulated normally in [rho 0] mutants. The results are consistent with a classical allosteric coeffector function for heme, although more indirect explanations are tenable. A role for the ROX1 gene product in transcriptional regulation can be inferred from the observation that there was no inhibition of ANB1 expression by heme in rox1 mutants. Judging from this epistasis the rox1 phenotype is not due to a defect in heme production; this would indicate that the ROX1 factor functions by mediating the effect of heme on transcription.

Oxygen equilibria of Octopus dofleini hemocyanin.

Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function.

A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm.

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.