Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolus aureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 104 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with nH values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme. H4folate; (±)-L-tetrahydrofolate

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila

Cytosolic nucleotidase II (cN-II) from Legionellapneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DatabaseThe coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers and . The accession number of GMP complexed LpcN-II is . Structured digital abstract andby() andby() Structured digital abstract was added on 5 March 2014 after original online publication]

Role of the loop L-4,L-5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L-4,L-5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L-4,L-5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L-4,L-5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the p-barrel 1 domain that harborst he GTP-bindings ite). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

The water effect on allosteric regulation of hemoglobin probed in water glucose and water glycine solutions

We have previously proposed a role of hydration in the allosteric control of hemoglobin based on the effect of varying concentrations of polyols and polyethers on the human hemoglobin oxygen affinity and on the solution water activity (Colombo, M. F., Rau, D. C., and Parsegian, V. A. (1992) Science 256, 655-659). Here, the original analyses are extended to test the possibility of concomitant solute and water allosteric binding and by introducing the bulk dielectric constant as a variable in our experiments. We present data which indicate that glycine and glucose influence HbA oxygen affinity to the same extent, despite the fact that glycine increases and glucose decreases the bulk dielectric constant of the solution. Furthermore, we derive an equation linking changes in oxygen affinity to changes in differential solute and water binding to test critically the possibility of neutral solute heterotropic binding. Applied to the data, these analyses support our original interpretation that neutral solutes act indirectly on the regulation of allosteric behavior of hemoglobin by varying the chemical potential of water in solution. This leads to a displacement of the equilibrium between Hb conformational states in proportion to their differential hydration.

Allosteric regulation of the primase (DnaG) activity by the clamp-loader (τ) in vitro

During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the τ subunit of the clamp-loader that loads the β clamp and interconnects the core polymerases on the leading and lagging strands. The τ–DnaB−DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the τ subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB–τ interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V τ mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of τ elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native τ protein. Thus changes in the τ–DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of τ are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by τ-interacting components of the replisome through the τ–DnaB−DnaG pathway.

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-l-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Å resolution crystal structure of the C trimer–PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer–PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.