Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection.

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.     

Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.     

Cooperative homotropic interaction of L-noradrenaline with the catalytic site of phenylalanine 4-monooxygenase

Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin (S = 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20 degrees C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of L-noradrenaline to phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h = 1.9); at pH 7.2 and 20 degrees C the rat enzyme binds about 0.5 mol L-noradrenaline/mol subunit with a half-maximal binding (S50) at 0.25 microM L-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate L-phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with L-phenylalanine also leads to a complete loss of the cooperativity of L-noradrenaline binding (h = 1.0). The many similarities in binding properties of the inhibitor L-noradrenaline and the activator/substrate L-phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(III) complex.     

The effect of chirality on serotonin receptor affinity.

α-Methylation of phenethylamines or tryptamine appears to have very little effect on serotonin (5-HT) receptor affinity when racemates are examined. Examination of resolved materials, using an isolated rat stomach fundus preparation, reveals that the R-isomers of DOM and MDA and the S-isomer of α-methlytryptamine possess a higher 5-HT receptor affinity than do their enantiomers. While S(+)-DOB possesses a lower affinity than its racemate, R(-)-DOB produces an appreciable agonistic response which interferes with the measurement of p_A2 values.     

Purification and properties of the arginine-specific carbamoyl-phosphate synthase from Saccharomyces cerevisiae.

The arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (Km = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.     

The binding of substrates and a product of the enzymatic reaction to glutathione S-transferase A.

The binding of substrates and a product to glutathione S-transferase A from rat liver was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrates glutathione and bromosulfophthalein as well as a product of glutathione and 3,4-dichloro-1-nitrobenzene, S-(2-chloro-4-nitrophenyl)glutathione, gave hyperbolic binding isotherms with a stoichiometry of 2 mol per mol of enzyme (i.e. 1 molecule per subunit). Glutathione (and glutathione disulfide) had an equilibrium (dissociation) constant for the binding of about 10 microM, whereas bromosulfophthalein and the product had equilibrium constants of about 0.5 microM. All ligands showed the same binding stoichiometry, and competition experiments involving unlabeled ligands indicated that glutathione and the glutathione derivatives were binding to the same site. Low affinity sites appeared to exist in addition to the specific high affinity sites (one per subunit) for all ligands tested. The binding studies are fully consistent with a steady state random kinetic mechanism for the enzyme.     

Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.     

The "regulatory" sulfhydryl group of Penicillium chrysogenum ATP sulfurylase. Cooperative ligand binding after SH modification; chemical and thermodynamic properties

ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S(50)=5.4 microM, n=1.0) but retained cooperativity of alpha-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K(D)=0.3 microM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6beta-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.     

Comparison of In vivo D-2 dopamine receptor binding of IBZM and NMSP in rat brain

In vivo biodistribution of S- and R-isomers of [¹²⁵I]IBZM in rats showed a significant initial brain uptake (3.20 and 2.67% dose/organ at 2 min, respectively). The wash-out from the brain was slower for the S-isomer. The striatum to cerebellum ratio for [¹²⁵I]S-IBZM decreased with an increasing dose of cold carrier or spiperone, suggesting that the brain uptake is stereospecific and saturable, and may be related to the binding of D-2 dopamine receptors. In a dual isotope digital autoradiography study [¹²⁵I]IBZM and [³H]NMSP(N-methylspiperone) show comparable regional cerebral distribution in rats.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S.

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive. The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin). Under these conditions approximately 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase

The allosteric transition of Escherichia coli aspartate transcarbamylase involves significant alterations in structure at both the quaternary and tertiary levels. On the tertiary level, the 240s loop (residues 230-245 of the catalytic chain) repositions, influencing the conformation of Arg-229, a residue near the aspartate binding site. In the T state, Arg-229 is bent out of the active site and may be stabilized in this position by an interaction with Glu-272. In the R state, the conformation of Arg-229 changes, allowing it to interact with the beta-carboxylate of aspartate, and is stabilized in this position by a specific interaction with Glu-233. In order to ascertain the function of Arg-229, Glu-233, and Glu-272 in the catalytic and cooperative interactions of the enzyme, three mutant enzymes were created by site-specific mutagenesis. Arg-229 was replaced by Ala, while both Glu-233 and Glu-272 were replaced by Ser. The Arg-229----Ala and Glu-233----Ser enzymes exhibit 10,000-fold and 80-fold decreases in maximal activity, respectively, and they both exhibit a 2-fold increase in the aspartate concentration at half the maximal observed velocity, [S]0.5. The Arg-229----Ala enzyme still exhibits substantial homotropic cooperativity, but all cooperativity is lost in the Glu-233----Ser enzyme. The Glu-233----Ser enzyme also shows a 4-fold decrease in the carbamyl phosphate [S]0.5, while the Arg-229----Ala enzyme shows no change in the carbamyl phosphate [S]0.5 compared to the wild-type enzyme. The Glu-272 to Ser mutation results in a slight reduction in maximal activity, an increase in [S]0.5 for both aspartate and carbamyl phosphate, and reduced cooperativity. Analysis of the isolated catalytic subunits from these three mutant enzymes reveals that in each case the changes in the kinetic properties of the isolated catalytic subunit are similar to the changes caused by the mutation in the holoenzyme. PALA was able to activate the Glu-233----Ser enzyme, at low aspartate concentrations, even though the mutant holoenzyme did not exhibit any cooperativity, indicating that cooperative interactions still exist between the active sites in this enzyme. It is proposed that Glu-233 of the 240s loop helps create the high-activity-high-affinity R state by positioning the side chain of Arg-229 for aspartate binding while Glu-272 helps stabilize the low-activity-low-affinity T state by positioning the side chain of Arg-229 so that it cannot interact with aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme-anthranilate-pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accommodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 microM) and phosphoribosylpyrophosphate (Km = 50 microM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 microM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod-Wyman-Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.     

Partial purification and properties of the isozymes of S-adenosylmethionine synthetase from sheep liver

Three forms of AdoMet synthetase were separated from sheep liver. The apparent molecular weights of the native isozymes were 122,000, 62,400 and 70,800 for the alpha-, beta 1- and beta 2-form, respectively and beta 1 was the predominant form. The alpha-form exhibited negative cooperativity with [S] 0.5 values of 31 microM for methionine and 62 microM for ATP; while the two beta-forms exhibited positive cooperativity with [S]0.5 values for methionine of 82 microM and 70 microM and those for ATP of 572 microM and 505 microM for the beta 1- and beta 2-form, respectively. Dimethylsulfoxide markedly stimulated the activities of the two beta-forms at low methionine concentrations. However, at high methionine levels, it inhibited the activity of the beta 2-form but not that of the beta 1-form. The effect of dimethylsulfoxide on the alpha-form was not significant. AdoMet was inhibitory at high concentrations. However, it had a slight stimulatory effect on the two beta-forms at low concentrations when methionine level was also low. These results suggest that AdoMet synthetase is a regulatory enzyme and the reaction rate in vivo can be directly influenced by substrate and product concentrations.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.