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The dynamic behavior of cyclic enzyme systems where two enzymes share a substrate or cofactor in a cyclic manner is investigated by computer simulation. The dynamic characteristics of cyclic enzyme systems display catastrophic behavior in response to specific changes in external input without assuming allosteric regulation.
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Several classes of centrally acting convulsant, depressant, anticonvulsant and anxiolytic drugs modulate GABAergic transmission. The postsynaptic receptor with which these drugs interact is an allosteric complex with distinct binding sites for GABA, benzodiazepines, picrotoxinin and related compounds. Convulsants which inhibit GABA transmission (except bicuculline) inhibit competitively the binding of dihydropicrotoxinin (DHP) or t-butylbicyclophosphorothionate (TBPT) to the picrotoxinin site and prevent the allosteric enhancing effect of depressant drugs on GABA and benzodiazepine binding. Depressant drugs give a mixed inhibition of TBPT binding. The possible topography of the picrotoxinin site and its relationship to convulsant/depressant drug action at the benzodiazepine-GABA receptor-ionophore complex is discussed.
Peptides can be transported across the blood-brain barrier by saturable transport systems. One system, characterized with radioactively labeled Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide), is specific for some of the small peptides with an N-terminal tyrosine, including Tyr-MIF-1, the enkephalins, beta-casomorphin, and dynorphin (1-8). Another separate system transports vasopressin-like peptides. The choroid plexus has at least one system distinguishable from those above that is capable of uptake and possibly transport of opiate-like peptides. The possibility of saturable transport of other peptides has been investigated to a varying degree. Specificity, stereo-specificity, saturability, allosteric regulation, modulation by physiologic and pharmacologic manipulations, and noncompetitive inhibition have been demonstrated to occur in peptide transport systems and suggest a role for them in physiology and disease.
Two high affinity phencyclidine (PCP) binding sites, labelled by [3H] 1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP), have been identified in guinea pig brain, with one site coupled to the N-methyl-D-aspartate (NMDA) receptor (site 1) and the other site associated with the dopamine reuptake carrier complex (site 2). In this study, PCP enhanced the dissociation of [3H]TCP from PCP site 1 and site 2, while (+)-MK801 only enhanced dissociation of [3H]TCP from PCP site 1. Although additional studies will be required to determine the exact mechanism(s) of these effects, these data demonstrate that the interactions of PCP with both site 1 and site 2 are more complex than previously appreciated.
GABA activates specific ion channels in post-natal cerebellar neurons in primary culture generating Cl- currents that can be recorded with the whole-cell patch-clamp technique. Evoked and spontaneous GABA-mediated synaptic activity can be recorded from cells kept in culture for a few days. Benzodiazepine and beta-carboline derivatives which bind with high affinity to the domains for allosteric regulation of GABA receptors facilitated and inhibited directly applied GABA responses and synaptically evoked Cl- currents recorded under voltage-clamp.
A functional N-type Ca2+ channel (omega-conotoxin GVIA receptor) has been purified from rabbit brain and shown to be composed of four subunits of molecular weights 230 K (alpha 1B), 160 K (alpha 2 delta), 95 K and 57 K (beta 3) [Witcher D. R., De Waard M., Sakamoto J., Franzini-Armstrong C., Pragnell M., Kahl S.D. and Campbell K. D. (1993) Science 261: 486-489]. These four subunits migrate on sucrose density gradients as a single complex and are identified by subunit specific polyclonal antibodies. Polyclonal antibodies against the purified receptor complex immunoprecipitate greater than 90% of the [125I]omega-conotoxin GVIA (omega-CgTx) binding sites in solubilized crude rabbit brain membranes. Furthermore, polyclonal antibodies affinity-purified against unique GST fusion proteins from two of the cloned subunits in the complex (alpha 1B and beta 3) specifically immunoprecipitated [125I]omega-CgTx binding sites and not [3H]PN200-110 binding sites. Analysis of [125I]omega-CgTx binding to the purified N-type Ca2+ channel demonstrated that the equilibrium binding was sensitive to increasing cation concentration. The IC50 for calcium and barium was 2.5 and 5 mM, respectively. [125I]omega-CgTx binding was not significantly reduced within 15 min after the addition of 50 mM barium. However, single channel analysis of the purified N-type Ca2+ channel preincubated with 10 microM omega-CgTx demonstrated that in the presence of 50 mM barium and 0.5 microM omega-CgTx, channel activity was detected but at a low open state probability (P < 0.10). These data suggest that the Ca2+ binding site(s) allosterically regulates the omega-CgTx binding site. Since the channel gating persisted in the presence of omega-CgTx, the omega-CgTx binding site may not be located within the pore of the channel and may be different from intra-pore Ca2+ binding sites.
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Activating mutations of p53 promote tumor progression. The mutant protein adopts a characteristic conformation, which lacks the growth suppressor function of wild-type p53. We show that mutant p53 can drive cotranslated wild-type p53 into the mutant conformation: a similar effect in vivo would block wild-type suppressor function with dominant negative effect. The cotranslational effect of mutant p53 on wild-type conformation depends upon interaction between nascent polypeptides and oligomerization of the full-length proteins. We also show that oligomers of p53 proteins can be induced to change conformation in a cooperative manner. Cell growth stimulation induces a similar conformational change in p53, and our present results indicate that this may involve allosteric regulation.
The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation.
Techniques are described for using blocking agents to distinguish between enzymes which are functional monomers and oligomers. To achieve this distinction the blocking agent must react exclusively at the active site with a stoichiometry of one mole of site per mole enzyme. The effect of the blocking agent on enzymatic activity in oligomers of n = 2 and 4 are described and the optimal degree of blocking is considered for tests of enzyme activity at saturating and less than saturating substrate concentrations. For saturating concentrations and a dimer the distinction between dimer and monomer is best observed with 50 per cent of sites blocked. For a tetramer the distinction is best made at higher degrees of blockade. The use of saturating substrate concentrations is thus limited to small oligomers. If nonsaturating substrate concentrations are used and normalized double reciprocal plots of the dependence of enzyme activity on substrate concentrations are made then the distinction between monomer and oligomer can readily be made for dimers, tetramers, and higher n-mers. The principles developed to distinguished monomeric from oligomeric enzymes are applied to published data obtained with the Ca Mg-ATPase of sarcoplasmic reticulum. Fluorescein isothiocyanate is the blocking agent. Plots of the published data support both the monomeric and tetrameric models for allosteric regulation with the preponderance of the data supporting the monomeric model.
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a soluble intracellular messenger formed rapidly after activation of a variety of cell-surface receptors that stimulate phosphoinositidase C activity. The initial response to Ins(1,4,5)P3 is a rapid Ca2+ efflux from nonmitochondrial intracellular stores which are probably specialized subcompartments of the endoplasmic reticulum, although their exact identities remain unknown. This initial response is followed by more complex Ca2+ signals: regenerative Ca2+ waves propagate across the cell, repetitive Ca2+ spikes occur, and stimulated Ca2+ entry across the plasma membrane contributes to the sustained Ca2+ signal. The mechanisms underlying these complex Ca2+ signals are unknown, although Ins(1,4,5)P3 is clearly involved. The intracellular receptor that mediates Ins(1,4,5)P3-stimulated Ca2+ mobilization has been purified and functionally reconstituted, and its amino acid sequence deduced from its cDNA sequence. These studies demonstrate that the Ins(1,4,5)P3 receptor has an integral Ca2+ channel separated from the Ins(1,4,5)P3 binding site by a long stretch of residues some of which form binding sites for allosteric regulators, and some of which are substrates for phosphorylation. In this review, we discuss the ligand recognition characteristics of Ins(1,4,5)P3 receptors, and their functional properties in their native environment and after purification, and we relate these properties to what is known of the structure of the receptor. In addition to regulation by Ins(1,4,5)P3, the Ins(1,4,5)P3 receptor is subject to many additional regulatory influences which include Ca2+, adenine nucleotides, pH and phosphorylation by protein kinases. Many of the functional and structural characteristics of the Ins(1,4,5)P3 receptor show striking similarities to another intracellular Ca2+ channel, the ryanodine receptor. These properties of the Ins(1,4,5)P3 are discussed, and their possible roles in contributing to the complex Ca2+ signals evoked by extracellular stimuli are considered.
Phosphofructokinase (PFK) from the malarial parasite Plasmodium berghei shows the following kinetic features: the more the pH is decreased, the more the enzyme is inhibited by ATP; in contrast to PFK from erythrocytes, this inhibition is less potent by two orders of magnitude; as in the red cell, fructose-6-phosphate (F6P) is a positive effector. Kinetic modelling of PFK from P. berghei has been performed by taking the pH-dependence of activity into regard, implicitly by the estimation of pH-dependent kinetic parameters for the inhibition by ATP and the activation by F6P and explicitly by the assumption of protonation-steps involved in allosteric regulation. By means of a novel procedure of model discrimination [D. Buckwitz and H.-G. Holzhütter: A new method to discriminate between enzyme-kinetic models. In: Application of Computational Methods in Medicine (Györi, I., ed.), Akademai, Budapest, in press] we have selected among several kinetic models the best rate equation which provides an adequate quantitative description of the kinetic behaviour of the enzyme in the relevant ranges of substrate concentrations and pH (5.8-7.6). It thus becomes clear how the highly increased glycolytic flux in malaria-infected cells could be affected through PFK.
According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively. It has been shown recently that the non-related antibiotics thiostrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism. Both drugs primarily block the allosteric transitions in either direction (Hausner et al. (1988) J. Biol. Chem. 263, 13103-13111). Here we show that the secondary effects of these antibiotics differ strikingly. When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition). The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound. The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes. We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population. These two populations probably reflect the P site in the pre- and post-translocational state, respectively.
The influence of fructose 1,6-bisphosphate and L-alanine on the kinetics of pyruvate kinase (ATP:pyruvate O2-phosphotransferase, EC 2.7.1.40) from Phycomyces blakesleeanus NRRL 1555 (-) was studied at pH 7.5. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenol pyruvate and Mg2+ were abolished and the velocity curves became hyperbolic. In the presence of L-alanine the positive homotropic cooperativity with respect to phosphoenol pyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by L-alanine, the antagonism between phosphoenol pyruvate and L-alanine also being evident. Inhibition has been found at high Mg2+ concentrations, compatible with the binding of the magnesium ions to an inactive conformational state of the enzyme. The data were analysed on the basis of the two-states concerted-symmetry model of Monod, Wyman and Changeux, and the parameters of the model were calculated. Phosphoenol pyruvate and fructose 1,6-bisphosphate appeared to show exclusive binding to the active conformational state (R), whereas magnesium ions bind preferentially, by a factor of 45, to the R state. L-Alanine binds more readily to the inactive T state of the enzyme.
The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated. Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents. This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide. The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate. The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+. Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme. The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme. The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed.