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NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits, designated IDH1 and IDH2. To determine the contribution of each subunit to regulation and catalysis, a conserved serine residue at the proposed active site of each subunit was mutated to alanine. This mutation in IDH1 resulted in a 6-fold decrease in Vmax and a decrease in cooperativity, but little change in S0.5 for isocitrate. The mutant IDH2, in contrast, exhibited a 60-fold decrease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate, but the cooperativity was unaffected. Responses to the allosteric modifier AMP also differed for the two mutant enzymes. The IDH1 mutant enzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibited an increase in isocitrate affinity in the presence of AMP similar to that observed with the wild-type enzyme. On the basis of these kinetic results, a model is presented which proposes that IDH1 functions as a regulatory subunit while IDH2 functions in catalysis. To determine if IDH1 or IDH2 alone is catalytically active, we also expressed the individual subunits in yeast strains in which the gene encoding the other subunit had been disrupted. Mitochondrial extracts from strains overexpressing solely IDH1 or IDH2 contained no detectable activity in the presence or absence of AMP. Gel filtration of these extracts showed that both IDH1 and IDH2 behaved as monomers, suggesting that the major subunit interactions within the octamer are between IDH1 and IDH2.
The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.
We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP). The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector. A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate. The mutant enzyme is also not activated by F1P or F6P. The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding. Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme. One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector. These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery. Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.
Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1. 41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1-7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands ('substrate-induced forms', numbered I-III) with different specific activities, dissociation constants of the enzyme-substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.
The activity of rabbit muscle pyruvate kinase (PK) is regulated by metabolites. Besides requiring the presence of its substrates, PEP and ADP, the enzyme requires Mg(2+) and K(+) for activity. PK is allosterically inhibited by Phe for activity. The presence of PEP or Phe has opposing effects on the hydrodynamic properties of the enzyme without an apparent change in secondary structure. In this study, the structural perturbation induced by ligand binding was investigated by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, the structural dynamics of PK was probed by H/D exchange monitored by FT-IR. Substrates and activating metal ions induce PK to assume a more dynamic structure while Phe exerts an opposite effect. In all cases there is no significant interconversion of secondary structures. PEP is the most efficient ligand in inducing a change in the microenvironments of both helices and sheets so much so that they can be detected spectroscopically as separate bands. These results provide the first evidence for a differential effect of ligand binding on the dynamics of structural elements in PK. Furthermore, the data support the model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the observed change in hydrodynamic properties represent the two extreme end states.
The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
We have previously shown that micromolar concentrations of GDP stimulate the GTP-mediated phosphorylation of p36, the alpha subunit of succinyl-CoA synthetase (SCS), in lysates prepared from Dictyostelium discoideum. In this study, we report that this phenomenon represents an enhanced catalytic capacity of SCS to form the phosphoenzyme intermediate. Low concentrations of GDP stimulate phosphoenzyme formation by either GTP, or succinyl-CoA and P(i). Under these conditions GDP enhances the apparent rate of phosphoenzyme formation but does not significantly alter the fraction of phosphorylated enzyme. This effect is retained during purification of the protein and is also observed with purified pig heart SCS, indicating that GDP directly alters the enzyme to enhance its rate of phosphorylation. Under these conditions, GDP does not function at the catalytic site, implying an allosteric regulation of SCS.
The kinetics of the absorption changes accompanying the photocycle of bacteriorhodopsin (BR) strongly depend on the intensity of the exciting short laser pulse. The decrease in the flash intensity dependence of the M kinetics after different extents of bleaching of the purple membranes by hydroxylamine proves the existence of a cooperative interaction between the photocycling BR molecules. The yield of the slow component of the M decay (M(s)) is a quadratic function of the extent of the fraction cycling. The slope of the relative weight of M(s) versus the fraction cycling is 0.5. This slope indicates a dimeric-like cooperative interaction, although the structural units of the purple membranes are the trimers of the BR molecules. For the most probable cooperative mechanism an asymmetric trimeric interaction is suggested, which accounts for the apparently dimeric features. A photocycling molecule may influence only one of its two neighbors in the trimer. From this asymmetric feature a deformative interaction is expected to be the cooperative mechanism, which would be an allosteric regulating mechanism in the purple membrane.
BACKGROUND: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes. RESULTS: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created. CONCLUSIONS: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation-a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.
The photoreceptor 3':5'-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory gamma subunit (Pgamma), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pgamma, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic alphabeta dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pgamma for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pgamma had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pgamma. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pgamma peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pgamma with the catalytic domain.
The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP. Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit. The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193. Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal. The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP. The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme. These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme.
To achieve high biological specificity, protein kinases and phosphatases often recognize their targets through interactions that occur outside of the active site. Although the role of modular protein-protein interaction domains in kinase and phosphatase signaling has been well characterized, it is becoming clear that many kinases and phosphatases utilize docking interactions - recognition of a short peptide motif in target partners by a groove on the catalytic domain that is separate from the active site. Docking is particularly prevalent in serine/threonine kinases and phosphatases, and is a versatile organizational tool for building complex signaling networks; it confers a high degree of specificity and, in some cases, allosteric regulation.
The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.
Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.
To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity.
Three artificial amino acids derived from l-serine by replacing the hydroxyl moiety with 1,4,7-triazacyclononane, 1,5,9-triazacyclododecane, and 1,4,7,10-tetraazacyclododecane, respectively, have been connected to the three arms of the tetraamine tris(2-aminoethyl)amine, Tren, to obtain tripodal ligands. They are able to bind up to four metal ions (like CuII and ZnII), three with the polyazamacrocycles and one with the Tren platform. Some of the ZnII complexes of these tripodal ligands proved to be good catalysts for the cleavage of the RNA model substrate 2-hydroxypropyl-p-nitrophenylphosphate (HPNP). Studies of the catalytic activity in the presence of increasing amounts of ZnII show that the complexes represent minimalist examples of metallocatalysts with cooperativity between the metal centers and allosteric control by a metal ion. The Tren binding site constitutes the allosteric regulation unit, while the three ZnII-azacrown complexes provide the cooperative, catalytic site. The allosteric role of the ZnII ion located in the Tren binding site was unambiguously demonstrated by studying the catalytic activity of a derivative unable to complex ZnII in that site. In this case, the cooperativity between the three ZnII ions bound to the peripheral azacrowns was totally suppressed. The kinetic analysis has shown that cooperativity is due to neither the occurrence of general-acid/general-base catalysis nor a decreased binding of the substrate because of the deprotonation of a water molecule bound to the complex but, rather, stabilization of the complexed substrate in its transformation into the transition state.
UMP is known to be an allosteric inhibitor of carbamyl phosphate synthetase, whereas IMP activates the enzyme. Surprisingly, dialdehyde UMP (prepared by periodate oxidation of UMP) was found to be a potent activator of the enzyme. Dialdehyde IMP, like IMP, produced activation. The corresponding dialcohol analogs of UMP and IMP (prepared by borohydride reduction of the dialdehyde analogs) had no effect on activity. These nucleotide interactions were further characterized by sedimentation velocity studies and by examination of the effects of inorganic phosphate on enzymatic activity. Although UMP promotes formation of an enzyme dimer, and IMP promotes formation of a tetramer (Powers, S. G., Meister, A., and Haschemeyer, R. H. (1980) J. Biol. Chem. 255, 1554-1558), the dialdehyde analogs of UMP and IMP both promote formation of mixed species. Low levels (less than 10 mM) of inorganic phosphate decrease the extent of activation by IMP, dialdehyde IMP, and dialdehyde UMP, but increase the extent of inhibition by UMP. The marked activation observed with dialdehyde UMP, and other considerations, suggest that the binding sites on the enzyme for IMP and UMP may overlap substantially. The findings also suggest that physiological levels of inorganic phosphate function in the modulation of the allosteric regulation of this enzyme by nucleotides.