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Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (Pi) and organophosphate (Po) compounds. Beyond synthetic Po compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring Po compounds to Pi, particularly with respect to solution versus surface speciation of Po hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L−1) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ± 0.8 µmol AMP, 35.7 ± 1.6 µmol ADP, and 10.9 ± 1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of Pi from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals.

Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from Synechocystis sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from Synechocystis sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 in vivo and in vitro, which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01–10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 μM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (–2.58 kcal·mol−1) and molecular distance (4.2 A). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.

Through transcriptional control of the powerful heat-shock or, proteotoxic stress, response (HSR/PSR), heat shock factor 1 (HSF1) preserves proteomic stability. Our studies reveal that HSF1, a physiological substrate for AMP-activated protein kinase (AMPK), suppresses this central metabolic sensor directly. By physically evoking conformational switching of AMPK, HSF1 blocks AMP binding to the γ subunits non-competitively, enhances the PP2A-mediated de-phosphorylation and impedes the LKB1-mediated phosphorylation of Thr172, as well as allosterically hinders ATP binding to the α subunits. These manifold regulations empower HSF1 to constrain AMPK activation in response to diverse cues, thereby sustaining cellular lipid content, serum lipid levels, liver and whole-body fat mass, as well as global protein cholesteroylation. In vivo, HSF1 antagonizes AMPK to drive the lipogenic phenotype and growth of melanomas, independently of its intrinsic transcriptional action. Thus, the physical AMPK-HSF1 interaction epitomizes an intriguing reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine.

Background: PstA is a cyclic di-AMP receptor and PII-like signal transduction protein. Results: Apo-PstA and complex crystal structures reveal a novel cyclic di-AMP-binding mode and induced conformational changes. Conclusion: PstA has a similar fold but distinct signal transduction properties from classic PII proteins, which function in nitrogen metabolism. Significance: Identification of common features allows for rational prediction of cyclic di-AMP-binding sites., Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA. Background: Cyclic di-AMP is an essential second messenger in eubacteria. Results: The c-di-AMP receptor DarA was identified in B. subtilis. The crystal structure and ITC data revealed the nucleotide specificity of DarA. Conclusion: DarA is a PII-like protein that undergoes conformational changes upon c-di-AMP binding. Significance: A novel PII-like protein is involved in c-di-AMP signaling.

One fundamental issue in the field of AMPs has been whether the action of AMPs produces pores in the membranes or disintegrates the membranes by the saturated surface binding of AMPs (the carpet model). In the absence of clear evidence, either mechanism seems to explain the phenomena of bacteria lysis. Even if the issue were resolved in the model membrane studies, it is not clear if the result of model membranes extends to bacterial membranes. Here we investigated the action of AMPs on E. coli spheroplasts. The absence of the outer membrane made it possible to observe the action of AMPs on the cytoplasmic membranes. Previously we found that the properties of the bacterial cell membranes are dominated by a membrane reservoir, significantly different from that of a GUV. Furthermore, these unique properties of bacterial membranes are metabolically maintained. We would like to know how the actions of AMPs on spheroplast membranes compare with the actions on GUVs. We compared the actions of human AMP LL-37 and melittin on spheroplasts and GUVs. We developed a fluorescence recovery after photobleaching (FRAP) technique to examine the dye leakage through the bacterial membranes. AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the responses of GUVs. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of FRAP showed that the fluxes of dye molecules in and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. The effects of LL37 and melittin are qualitatively the same.

This paper demonstrates and systematically characterizes the enrichment of biomolecular compounds using aptamer-functionalized surfaces within a microfluidic device. The device consists of a microchamber packed with aptamer-functionalized microbeads and integrated with a microheater and temperature sensor to enable thermally controlled binding and release of biomolecules by the aptamer. We first present an equilibrium binding-based analytical model to understand the enrichment process. The characteristics of the aptamer-analyte binding and enrichment are then experimentally studied, using adenosine monophosphate (AMP) and a specific RNA aptamer as a model system. The temporal process of AMP binding to the aptamer is found to be primarily determined by the aptamer-AMP binding kinetics. The temporal process of aptamer-AMP dissociation at varying temperatures is also obtained and observed to occur relatively rapidly (< 2 s). The specificity of the enrichment is next confirmed by performing selective enrichment of AMP from a sample containing biomolecular impurities. Finally, we investigate the enrichment of AMP by either discrete or continuous introduction of a dilute sample into the microchamber, demonstrating enrichment factors ranging from 566 to 686×, which agree with predictions of the analytical model.

In mammals, 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the gamma-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine beta-synthase domains so far. Site E is located in a small cavity existing between the alpha-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in gamma-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.

With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l with an IC50 value of 0.013 μM against human FBPase. An X-ray crystallographic study revealed that 8l bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated octameric enzyme composed of two types of homologous subunits designated IDH1 and IDH2. Based on sequence comparisons and structural models, both subunits are predicted to have adenine nucleotide binding sites. This was tested by alanine replacement of residues in putative sites in each subunit. Targets included adjacent aspartate/isoleucine residues implicated as important for determining cofactor specificity in related dehydrogenases and a residue in each IDH subunit in a position occupied by histidine in other cofactor binding sites. The primary kinetic effects of D286A/I287A and of H281A replacements in IDH2 were found to be a dramatic reduction in apparent affinity of the holoenzyme for NAD(+) and a concomitant reduction in V(max). Ligand binding assays also showed that the H281A mutant enzyme fails to bind NAD(+) under conditions that are saturating for the wild-type enzyme. In contrast, the primary effect of corresponding D279A/D280A and of R274A replacements in IDH1 is a reduction in holoenzyme binding of AMP, with concomitant alterations in kinetic and isocitrate binding properties normally associated with activation by this allosteric effector. These results suggest that the nucleotide cofactor binding site is primarily contributed by the IDH2 subunit, whereas the homologous nucleotide binding site in IDH1 has evolved for regulatory binding of AMP. These results are consistent with previous studies demonstrating that the catalytic isocitrate binding sites are comprised of residues primarily contributed by IDH2, whereas sites for regulatory binding of isocitrate are contributed by analogous residues of IDH1. In this study, we also demonstrate that a prerequisite for holoenzyme binding of NAD(+) is binding of isocitrate/Mg(2+) at the IDH2 catalytic site. This is comparable to the dependence of AMP binding upon binding of isocitrate at the IDH1 regulatory site.

AMP, an allosteric inhibitor at neutral pH, activates snake muscle fructose 1,6- bisphosphatase at pH 9.2. The activation is virtually unique for the snake muscle enzyme: activation was not observed for the enzymes from either human and rabbit liver or porcine kidney. The activation is Mg2+-dependent but was not observed until the concentration of Mg2+reaches 1 mM. It is known that subtilisin, trypsin, or lysosomal proteases hydrolyse the N-terminal loop of fructose-1,6-bisphosphatase in the vicinity of amino acid residue 60 generating a form of the enzyme with a pH optimum at 9.2. In the presence of AMP, the pH profile of the native snake muscle enzyme resembles that of the alkaline form and modification of the highly reactive sulfhydryl group abolishes AMP activation. The fact that AMP has a dual function at different pH levels suggests that pH might be an important factor in regulating the activity of the enzyme upon binding of AMP at the allosteric site. Indeed, the mode of AMP binding to the allosteric site may differ at neutral and alkaline pH levels. A residue that ionizes with a pKaof 8.9 might be involved in this process.

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 → Ala, Glu-192 → Gln, and Asp-187 → Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s−1 for wild-type enzyme to 15 s−1 and 13 s−1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 → Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.

We examine structural aspects of the allosteric transition of pig kidney fructose-1,6-bisphosphatase (Fru-1,6-Pase) by analyzing the X-ray structures of the R and T form enzymes. The results show a hierarchical structural change during the R to T transition. Upon binding of AMP, a cascade of structural changes occurs starting from the AMP site: expansion of the AMP site, local conformational changes of helices H1 and H2, independent rotations and translation of helices H1, H2 and H3 (and loops connecting them), reorganization of the AMP domain as a whole and its 1.9 degrees rotation relative to the fructose-1,6-bisphosphate domain, and conformational changes at the C1-C2 and C1-C4 interfaces leading to the quaternary conformational change of a 17 degrees rotation between dimers. The AMP inhibition results from the relative movement between the AMP and FBP domains which distorts the active site during the transition by shifting the metal binding sites to unfavourable positions. Communication that ensures cooperativity during R to T transition relies on changes in positions of helices H1, H2 and H3, loops 127-131, 168-170 and 187-192, and on N-terminal residues. All of these features are close to the C1-C4 and symmetry equivalent C2-C3 interfaces and the relatively small C1-C3 interface of the T form. These secondary structures form the framework along which structural changes due to AMP binding can propagate to other parts of the monomers as well as across monomer interfaces. Future dynamics studies may be useful to analyze initiation, propagation and completion of the quaternary conformational change of Fru-1,6-Pase upon AMP binding. Also, site directed mutagenesis experiments are expected to provide more detailed descriptions of the importance of each of the residues that has been identified here in the proposed mechanisms.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

The topological location of the nucleotide substrate binding environments on adenylate kinase has been explored with the fluorescent molecule [4-benzoyl]benzoyl-1-amidofluorescein (BzAF) and the nucleotide analog 3'-O-[4-benzoyl]benzoyl-ATP (BzATP), which are site-directed photoaffinity probes that bind covalently at the individual nucleotide sites. The MgBzATP substituted for MgATP as a substrate for this enzyme, whereas BzAF, which is neither a substrate nor a nucleotide, behaved as a competitive inhibitor for each nucleotide site independently. BzAF could be directed specifically to either of the nucleotide sites by near saturation of the second site with its natural nucleotide substrate. Using this second site blocking approach, each nucleotide site, in turn, could be protected competitively from BzAF-induced photoinhibition by the presence of its natural substrate. This strategy showed that under photolysis, the Kd(BzAF) of 0.1 mM (for the MgATP site) and 0.2 mM (for the AMP site) were nearly identical with the Km(app) values determined for MgATP and AMP, respectively. Pseudo first-order photolysis kinetics with [3H]BzAF revealed covalent binding stoichiometries for full inhibition of 1 mol of probe/mol of enzyme at either site. Thus, we prepared the binary complex ([3H]BzAF-enzyme) by photo-labeling the MgATP site-blocked enzyme with [3H]BzAf to 1:1 molar stoichiometry. Tryptic digestion followed by partial sequencing of the [3H]BzAF-labeled enzyme disclosed that BzAF was bound specifically within the peptide span Gly64-->Arg77. This amino acid domain therefore probably constitutes the neighborhood identifiable with AMP binding. Furthermore, we also achieved double photocovalent labeling of adenylate kinase with both photoprobes, by first cross-linking with approximately 1 mol of MgBzATP, followed by approximately 0.9 mol of [3H]BzAF, thus generating a ternary covalent complex. Comparison of the fluorescence of the binary species and the ternary (BzATP-enzyme-[3H]BzAF) complex revealed altered fluorescence emission profiles, which could indicate that a conformational change occurs during formation of the ternary (or transition state) complex.

Nucleotide-depleted mitochondrial F1-ATPase (F1[0,0]) is inhibited by the diadenosine oligophosphate compounds, AP4A, AP5A, and AP6A (where APxA stands for 5',5'-diadenosine oligophosphates having a chain of x phosphoryl groups linking the two adenosine moieties). When F1[0,0] is preincubated with these compounds and then assayed for ATP hydrolysis activity under conditions that normally allow turnover at all three catalytic sites, the maximal level of inhibition observed is 80%. However, when assayed at lower ATP concentrations under conditions that allow simultaneous turnover at only two of the three sites, no inhibition is observed. A decrease in the number of phosphoryl groups that links the adenosine moieties to less than 4 (AP3A, AP2A) converts the compound to an activator of ATP hydrolysis, similar in effect to that obtained when one mol of ADP or 2-azido-ADP binds at a catalytic site on F1[0,0]. Inhibition by the compounds requires the presence of at least one vacant noncatalytic site. Evidence is provided that the probes also interact with a catalytic site. The stoichiometry for maximal inhibition by AP4A is 0.94 mol/mol of F1. The data presented support a model for the structure of nucleotide-binding sites on F1 that places catalytic and noncatalytic sites in close proximity in an orientation analogous to the ATP and AMP binding sites on adenylate kinase. Inhibition of the enzyme by the dinucleotide compounds can be explained by the cross-bridging of one of the catalytic sites to a noncatalytic site in analogy to the inhibition of adenylate kinase by AP5A. The residual capacity for bi-site catalysis indicates that the second and third catalytic sites remain catalytically active.

Advances in photochemical generation of “caged” biochemical intermediates ( 1) have made it possible to examine kinetic events that are too fast or inappropriate for stopped-flow methods. We have done experiments to test whether the chemistry of photochemical generation of organic phosphates can be used in the presence of spinlabeled macromolecules. It was a concern that free-radical intermediates generated in the photolysis might react with the nitroxide groups. Specifically, we have examined the known conformational change of spin-labeled phosphorylase b when it binds AMP ( 2). The effector, AMP, was generated photochemically from its 1 ( 2-nitrophenyl ) ethyl ester. For the test, the photolysis was done with a mercury arc lamp. The results suggest that it should be feasible to conduct experiments in the time-resolved mode using EPR detection with spin labels and laser-induced photolysis of caged intermediates to initiate events. Phosphorylase b is a dimer that exhibits cooperativity between subunits (3). The protein can be labeled with an iodoacetamide spin label at cysteine-3 17 (4). The factors that contribute to altered EPR signals when spin-labeled phosphorylase b from rabbit muscle interacts with effecters have been examined in detail (2). Phosphorylase b is activated when AMP binds to the activator site and the EPR signal of the spinlabeled protein changes on AMP binding. In addition, there is a different nucleoside inhibitor site to which adenine, adenosine, and other inhibitors bind. Pyridoxal 5’phosphate serves as a cofactor. Caged AMP was prepared (5) by DCC coupling of I-(2-nitrophenyl)ethanol to pyridinium AMP in dry pyridine. After complete removal of pyridine, caged AMP was separated from starting materials on a DEAE-A25 Sephadex column (bicarbonate form, pH 7.0, 0 to 0.26 M triethylammonium bicarbonate linear gradient). Thinlayer chromatography on silica gel plates in a solvent system of isopropanol, ammonium hydroxide, water (7, 2, 1 v/v) gave a spot with Rf 0.82 which gave, after photolysis, a spot of Rf 0.33, identical to that of AMP. Microanalysis was consistent with the formula of caged AMP * 2.5 H20 and inconsistent with the formulas of starting materials. Phosphorylase b was prepared with modifications of the procedure of Fischer

Disorder-to-order transitions in proteins can play critical roles in regulating function. Corepressor bio-5'-AMP binding to the E. coli biotin repressor promotes homodimerization that is a prerequisite to sequence-specific DNA binding and resulting transcription repression. Folding of a loop segment comprised of residues 211-222 in the corepressor binding site is required to obtain the full −4.0 kcal/mole energetic coupling between bio-5'-AMP binding and dimerization. Several additional loops, some of which are disordered in the unliganded monomer but folded in the liganded dimer, participate directly in the homodimer interface. Alanine scanning mutagenesis has demonstrated the functional significance of these loops for dimerization. Replacement of a glycine at position 142 in loop 140-146 with alanine results in complete abolition of coupling of corepressor binding to dimerization. Consistent with solution measurements and in contrast to wild type BirA, the structure of this variant solved by x-ray crystallography reveals a monomeric liganded protein. Moreover, the alanine replacement results in adoption of an alternative conformation by the 140-146 loop. Furthermore, distinct from wild type corepressor-bound BirA, both a neighboring dimer interface loop comprised of residues 193-199 and the 211-222 loop that is located 33 A away from the alanine replacement are disordered in the liganded variant. These results support a model for energetic coupling between ligand binding and dimerization in which distant disorder-to-order transitions are communicated through the folded protein core.

Equilibrium dialysis and isothermal microcalorimetry experiments have been carried out to characterize the thermodynamics of the binding of AMP to glycogen phosphorylase b (EC 2.4.1.1) at pH 6.9 over the temperature range of 25-35 degrees C. Thermal titrations were performed at each temperature in various buffer systems, which have afforded the calculation of the number of protons exchanged when the AMP binds to each site in the protein. Thermodynamic parameters were obtained for the binding of AMP to the two nucleotide and the two inhibitor sites of the dimeric enzyme. The former show positive cooperativity while the latter behave as independent binding sites. A positive delta Cp value was obtained for the AMP binding to the two N sites (1.3 and 1.4 kJ K-1 mol-1), while the delta Cp was negative for the binding to the I sites (-1.9 kJ K-1 mol-1). The application of Sturtevant's method to our data (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240) and their comparison with a similar analysis undertaken with phosphorylase a (Mateo, P. L., Gonzalez, J. F., Baron, C., Lopez-Mayorga, O., and Cortijo, M. (1986) J. Biol. Chem. 261, 17067-17072) has opened the way to some understanding of the thermodynamics of the allosteric transition in the protein.

Fructose 1,6-bisphosphatase has been isolated in homogeneous form from rat liver by a simple and convenient procedure, including adsorption on carboxymethyl cellulose and substrate elution. It is a tetrameric protein, with molecular weight of approximately 140,000. The amino acid composition shows some similarity to that of the rabbit liver enzyme, with a blocked NH 2 -terminus and leucine, rather than alanine, as the COOH-terminal residue. The purified enzyme contains no tryptophan. It requires Mg 2+ or Mn 2+ and is activated by NH 4 + , which also decreases the affinity for both substrate and divalent cations. In the absence of chelating agents, the pH optimum is 8.0, but this is shifted to 7.0, 7.2, and 7.5 on addition of EDTA, histidine, and citrate, respectively. Ca 2+ is a potent inhibitor and appears to compete for the Mg 2+ -binding site. The rat liver enzyme is inhibited by AMP in a cooperative manner, with a Hill coefficient of about 2. Saturation is reached when approximately 2 mol of AMP are bound per mole of enzyme, but no AMP binding is observed in the absence of the substrate. Antibody to the purified enzyme, produced in the rabbit, reacts with fructose 1,6-bisphosphatase in rat liver and rat kidney with equal affinity. It also reacts with rat muscle fructose 1,6-bisphosphatase, with 1/100th the affinity.

The fluorescent probe 1-anilino-8-naphthalene sulfonate, when bound by the inactive form of phosphorylase b, behaves as if distributed between two different binding sites, whose bound dye emits fluorescence with decay times of 19 and 8 nanosecs. In 0.1 M glycylglycine, pH 7.0, the addition of the allosteric activator adenosine-5′-phosphate or the substrate glucose-1-phosphate results in the progressive loss of the component of longer decay time. At intermediate levels of activator the decay curves could be represented as the sum of the weighted contributions of two species, corresponding to those prevailing in the absence of activator and in excess activator. The activity of glycogen phosphorylase b, which is one of the most extensively studied allosteric enzymes, is controlled by the allosteric activator AMP (1, 2). Binding studies have shown that the binding of AMP in the absence of substrate is strongly cooperative, with a value of the Hill coefficient of 1.7 (2). Binding and kinetic studies have indicated that strong heterotropic cooperativity exists between the combination of the enzyme with AMP and either of the substrates glucose-1-phosphate and inorganic phosphate (1, 2, 3, 4). Phosphorylase b (molecular weight 185,000) consists of two equivalent subunits of molecular weight 92,500. Each subunit contains a single strong binding site for AMP(1, 2, 3). The AMP-induced activation of phosphorylase b has usually been attributed to a conformational transition, AMP being preferentially bound by the active form (1). The high degree of cooperativity of AMP binding has been attributed to the simultaneous conversion of both subunits to the active conformation upon the binding of a molecule of AMP by one subunit (1, 2). The existing kinetic studies have been interpreted as indicating that the binding of substrate likewise favors the transition to the active form, although differences of opinion exist as to the number and equivalence of the active states (1, 2, 5). However, a basic weakness of the model described above is the paucity of direct physical evidence for a conformational change. In recent years a rapid evolution of technique has made it possible to monitor the time decay of fluorescence directly (6). The technique of nanosecond fluorometry has been applied to a number of systems with the objective of analyzing complex decay curves or detecting excited state reactions (7). With the development of programs for analyzing multicomponent decay curves in terms of the lifetimes and amplitudes of the individual emitting species (8) it has become possible to apply nanosecond fluorometry to the problem of monitoring conformational transitions of proteins, and in particular, to the activator - or substrate - induced transformations of allosteric proteins. In general, when emission occurs from a collection of sources with different decay times, we have for the fluorescence intensity i (t) as a function of time, t: (1) i(t) = ∑ j α j , e , -t τ j where αj and τj are the amplitude and decay time, respectively, corresponding to the jth component. If the number of components is small, the experimental curves of i(t) versus t may, in principle, be analyzed by the method of moments to yield the set of values of αj and τj. In this report, we shall describe the results of the application of nanosecond fluorometry to the phosphorylase b system, using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). This probe, which is virtually non-fluorescent in aqueous solution, acquires an intense fluorescence when bound by a non-polar site on a protein (9, 10). In an earlier report, Seery and Anderson have found that the fluorescence intensity of ANS bound to phosphorylase b is sharply reduced in the presence of AMP or substrates. From parallel equilibrium dialysis studies, it was concluded that the greater part of the effect arose from the dissociation of ANS, although it was not possible to demonstrate a quantitative correlation (11). For this reason, it was not possible to make any statement as to the quantum yield or excited lifetime of the residual bound dye.

Rabbit muscle phosphorylase b reacts with the phosphate-like reagent potassium ferrate, K2FeO4, a potent oxidizing agent. The reaction results in inactivation of the enzyme and abolition of the ability of the enzyme to bind 5'-AMP. Activating and nonactivating nucleotides which bind at the 5'-AMP binding site such as 5'-AMP, 2'-AMP, 3'-AMP, and 5'-IMP substantially protect the enzyme from inactivation by ferrate. One to two residues of tyrosine and approximately 1 residue of cysteine are modified by ferrate under the conditions employed. Tyrosine is protected by 5-AMP, whereas cysteine is not. The tyrosine modification is suggested as the inactivating chemical reaction. The location of the inactivating reaction is suggested to be in or near the 5'-AMP binding site. The structural and chemical properties of ferrate ion are discussed and compared to those of phosphate. Ferrate ion may be a reagent useful for phosphate group binding site-directed modification of proteins.

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.

The effects of AMP, fructose 6-phosphate (Fru-6-P), fructose 2,6-bisphosphate (Fru-2,6-P2), and paramagnetic ions on the aromatic region of the proton nuclear magnetic resonance (NMR) spectrum of rabbit liver fructose-1,6-bisphosphatase have been investigated at 300 MHz. Two well resolved peaks in this region of the NMR spectrum are assigned to the protons from the aromatic ring of a tyrosyl residue of the enzyme by chemical modification with tetranitromethane and by nuclear Overhauser effects. Nitration of the tyrosyl residue causes desensitization of the enzyme to AMP inhibition as well as the loss of activity. In the presence of AMP during the modifications, 1 tyrosyl residue could be protected, presumably the one observed by NMR. Binding of AMP, an allosteric inhibitor of the enzyme, to rabbit liver fructose-1,6-bisphosphatase leads to an upfield shift of the tyrosyl proton signals in the NMR spectrum. No chemical shift or line broadening could be detected in the presence of the paramagnetic manganous ion, Fru-2,6-P2, or Fru-6-P. The negative intramolecular nuclear Overhauser effect from the ribose H2' proton to the adenine H8 proton of AMP suggested that AMP binds to the enzyme with an anti conformation about the glycosidic bond. The failure to observe intermolecular nuclear Overhauser effects between the tyrosyl residue and the protons of AMP indicates that the distances between them are greater than 4 A. On the basis of these observations, it is suggested that the AMP-related tyrosyl residue may be close to the AMP binding site, but it is not directly involved in ligand binding. Rather, the protection of this tyrosyl residue by AMP as observed by chemical modification experiments may well be due to a conformational change that results from covalent modification of the enzyme.

To elucidate further how cyclic adenosine 3' : 5'-monophosphate (cyclic AMP) and the cyclic AMP receptor are able to stimulate gal mRNA synthesis we have utilized an antiserum specific for cyclic AMP receptor and also cyclic GMP as inhibitors of cyclic AMP and cyclic AMP receptor action. When cyclic AMP, cyclic AMP receptor, DNA, and RNA polymerase are incubated together a rifampicin-resistant preinitiation complex is formed and, upon the addition of missing ribonucleoside triphosphates or Mg2+, gal mRNA synthesis occurs. If the preinitiation complex is formed in the absence of nucleoside triphosphates the antiserum is able to dissociate the complex. If the complex is formed in the presence of nucleoside triphosphates (no Mg2+), it is converted to a more stable form which is not reversed by the addition of antiserum. Cyclic GMP can prevent the formation of the preinitiation complex. However, we find that once the preinitiation complex has been formed cyclic GMP does not inhibit cyclic AMP receptor-cyclic AMP-dependent transcription, suggesting that RNA polymerase stabilizes cyclic AMP receptor-cyclic AMP binding to the gal promoter region.

Acinar cells from guinea pig pancreas possess two distinct protein kinase activities. Cyclic GMP-dependent kinase elutes as a single peak on diethylaminoethyl (DEAE)- cellulose chromatography, is not inhibited by protein kinase inhibitor, and has a greater affinity for cyclic GMP (half-maximal activation at 20 nM) than for cyclic AMP (half-maximal activation at 100 nM). Cyclic AMP-dependent kinase elutes as two peaks on DEAE-cellulose chromatography, is inhibited by protein kinase inhibitor, and has a greater affinity for cyclic AMP (half-maximal activation at 20 nM) than for cyclic GMP (half-maximal activation at 7 micrometer). Binding of cyclic 3H-nucleotides to the enzyme preparation was rapid, specific, temperature-dependent, and reversible, and there was a close correlation between the ability of a particular cyclic nucleotide to inhibit binding of cyclic 3H-nucleotide and its ability of a particular cyclic nucleotide to inhibit binding of cyclic H-nucleotide and its ability to activate protein kinase. Binding of cyclic 3H--nucleotide could not be described as a simple bimolecular reaction and native cyclic nucleotides accelerated the dissociation of bound, labeled cyclic nucleotide. Vasoactive intestinal peptide or secretin, each of which increases cellular cyclic AMP, caused endogenous activation of protein kinase and inhibition of cyclic [3H]AMP binding but did not alter bindings of cyclic [3H]GMP or cyclic [3H]AMP.

The binding of AMP and ADP to specific sites on the human platelet membrane have been compared. Although the two nucleotides bound to the platelet membrane with similar affinities (Ka ⋍ 0.4 × 10 6 M −1 ), AMP binding was more rapid than ADP binding and involved a reduced number of binding sites. AMP competitively inhibited ADP binding by reacting with approximately half of its binding sites. Thus, the binding of AMP to specific sites in human platelet membrane provides a tool to differentiate two different types o£ ADP binding sites with similar affinities. AMP does not inhibit membrane nucleoside diphosphokinase, the enzyme which converts ADP into ATP when intact washed platelets or isolated platelet membranes are incubated with ADP in presence of divalent cations.

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.

A model of the polypeptide backbone of the dimer of glycogen phosphorylase a (EC 2.4.1.1) was built from a 3 A resolution electron density map derived from x-ray diffraction analysis of native tetragonal crystals and two heavy atom isomorphous replacement derivatives. Each identical subunit of the dimer has a compact shape with overall dimensions of 85 X 75 X 55 A and is tightly associated with its 2-fold symmetry related subunit. There are three major excursions of the polypeptide chain of one monomer across the 2-fold axis to make extensive contacts with the other subunit. The active site, of which there are two per dimer, is shared between the two subunits at their interface and comprises a pocket-like region within a "V"-shaped framework of two alpha helices. Within this region are found the binding sites for the substrates, glucose-1-P and arsenate, a competitive inhibitor, UDP-glucose, and the allosteric effector, AMP. The site of metabolic control, Ser-14 phosphate, is hydrogen-bonded to a side chain on the outside of one of the alpha helices forming the active site and is 15 A from the AMP binding site. Maltoheptaose, a glycogen analogue and substrate for these enzymatically active crystals, binds in a second region of interest. Even at concentrations above its Km, when binding is sufficiently tight that all seven glucose moieties may be discerned, the closest of these is 25 A from the glucose-1-P binding site. We suggest that this polysaccharide binding site may represent a storage site whereby phosphorylase is bound to the glycogen particle in the muscle cell. The polypeptide chain in a third region has the same topological structure as has been observed for the nucleotide binding domains in the dehydrogenases. Adenine or adenosine (but not AMP) bind here in a position similar to the adenine ring of NAD in the dehydrogenases while glucose binds 17 A away in an interior crevice near the center of the monomer.

Kinetic studies with ADP-glucose synthase show that 1,6-hexanediol bisphosphate (1,6-hexanediol-P2) is an effective activator that causes the enzyme to have a higher apparent affinity for ATP- and ADP-glucose than when fructose-1,6-P2 is the activator. Furthermore, in the presence of 1,6-hexanediol-P2, substrate saturation curves are hyperbolic shaped rather than sigmoidal shaped. CrATP behaves like a nonreactive analogue of ATP. Kinetic studies show that it is competitive with ATP. CrATP is not a competitive inhibitor of ADP-glucose. However, the combined addition of CrATP and glucose-1-P inhibits the enzyme competitively when ADP-glucose is the substrate. In binding experiments, CrATP, ATP, and fructose-P2 appear to bind to only half of the expected sites in the tetrameric enzyme, while ADP-glucose, the activators, pyridoxal-P and 1,6-hexanediol-P2, and the inhibitor, AMP, bind to four sites/tetrameric enzyme. Fructose-P2 inhibits 1,6-hexanediol-P2 binding, suggesting competition for the same sites. Glucose-1-P does not bind to the enzyme unless MgCl2 and CrATP are present and binds to four sites/tetrameric enzyme. Alternatively, CrATP in the presence of glucose-1-P binds to four sites/tetrameric enzyme. Thus, there are binding sites for the substrates, activators, and inhibitor located on each subunit and the binding sites can interact homotropically and heterotropically. ATP and fructose-P2 binding is synergistic showing heterotropic cooperativity. ATP and fructose-P2 must also be present together to effectively inhibit AMP binding. A mechanism is proposed which explains some of the kinetic and binding properties in terms of an asymmetry in the distribution of the conformational states of the four identical subunits.

Prostaglandins at mM concentration are able to induce a small activation to rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) in the absence of AMP. The extent of this activation depends on the nature of the molecular structure of prostaglandins. Saturated and unsaturated higher fatty acids were unable to substitute for prostaglandins. The main findings of our studies can be summarized as follows: (1) Prostaglandins inhibit the AMP-induced activation of phosphorylase b. (2) Modification of the AMP binding site with 2,3-butanedione could not inhibit the activity induced by prostaglandins. (3) Enzyme activation by prostaglandins is stimulated by spermine. (4) Phosphorylase b activation by prostaglandins and the observed stimulation of this activation by spermine were found to be temperature dependent. (5) Prostaglandins affect the quaternary structure of phosphorylase b inducing a partial enzyme tetramerization which is enhanced in the presence of spermine. The extent of this tetramerization is temperature dependent.

At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P 2 -ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that thep K value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH 2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP ( I 0.5 ), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.

Kinetic studies with adenylate deaminase have been performed by stopped flow methods at 20 degrees C in 0.01 M imidazole/HCl, pH 6.5. The data were analyzed using either the whole time course of the reaction or the initial portion of the full time course. At low KCl concentrations, activation by the product IMP complicates any interpretation. In the presence of 0.15 M KCl, the results are interpreted in terms of three types of purine nucleotide binding sites: an active site, an inhibitory site which appears to be relatively specific for nucleoside triphosphates, and an activating site which shows relatively little specificity for nucleoside phosphates. Nucleotide binding to the activating site weakens binding to the inhibitory site. Sigmoidal kinetic data observed as a function of AMP in the presence of the inhibitor GTP are interpreted in terms of AMP binding to the activating site and weakening GTP binding. A fragment of myosin, subfragement-2, which has previously been shown to form a tight complex with adenylate deaminase (Ashby, B., and Frieden, C. (1977) J. Biol. Chem. 252, 1869--1875) activates the deaminase reaction only slightly. Complex formation, however, makes the reaction less susceptible to inhibition by GTP, although high levels of this nucleotide will disrupt the complex. In the presence of GTP or GTP plus subfragment-2, hysteretic effects are observed.

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.

1. 1. The inhibition of bands 5 and 1 isoenzymes of porcine lactate dehydrogenase by salicylate was investigated kinetically. 2. 2. These studies indicated that up to two molecules of salicylate could combine with the active site of the free band 5 isoenzyme, and that one of the two salicylate molecules competes with its AMP binding site. 3. 3. It is concluded that salicylate competes with the AMP moiety of NAD+ in this enzyme.

The binding of AMP to rabbit muscle glycogen phosphorylase a (EC 2.4.1.1.) has been studied by equilibrium dialysis and isothermal microcalorimetry at pH 6.9 over a temperature range of 25 degrees C to 35 degrees C. Thermal titration experiments were carried out in various buffer systems. We have found by these methods that a certain number of protons are released when the protein binds to the ligand and are taken up by the buffer. The tetramer of phosphorylase a has been shown to have four equal and independent, non-cooperative binding sites for AMP at 25 degrees C, 30 degrees C, and 35 degrees C; these sites can be assigned to the so-called nucleotide or, activator, sites in the protein. The binding constants together with the changes in Gibbs energy, enthalpy, and entropy per site for the AMP binding were calculated at each temperature. A negative delta Cp value of -2.3 +/- 0.2 J K-1 (AMP bound)-1 was obtained for this binding process. The hydrophobic and vibrational contributions of the heat capacity and entropy changes have been resolved by the method described by Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). From this analysis, it appears that the binding is, in all cases, enthalpy-driven, the two entropic contributions, hydrophobic and vibrational, having opposing effects.

Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.

Kinetic studies of the inhibition of rabbit muscle phosphorylase a D-glucan:orthophosphate alpha-D-glucosyltransferase (EC 2.4.1.1) by various glucose derivatives indicate that glucose derivatives in the chair conformation (glucose, 2-deoxyglucose, glucosyl fluoride, and 1-O-methylglucose) stabilize the "T" form of phosphorylase a, whereas those in the half-chair conformation (1,5-gluconolactone and glucal) bind to the "R" form. Derivatives which stabilized the T form were generally competitive inhibitors of phosphate binding, were synergistic with caffeine, and weakened AMP binding. Derivatives which bound to the R form were noncompetitive with respect to Pi in the presence of AMP. The mechanism of inhibition of the R form by gluconolactone and glucal appears to depend upon the presence or absence of AMP. In the presence of the nucleotide, gluconolactone is probably a linear mixed-type inhibitor. However, in the absence of AMP, simple linear uncompetitive inhibition was apparent. Since gluconolactone is thought to be an analogue of the substrate transition state of the phosphorylase reaction, a major function of nucleotide in activating phosphorylase may be the formation of the transition state binding pocket in the binary enzyme-glycogen complex. In the absence of AMP, glucal was competitive with respect to phosphate. However, in the presence of nucleotide, simple intersecting linear noncompetitive inhibition was observed. The results indicate that a second function of AMP is the formation of the phosphate binding pocket in the enzyme-glucal-glycogen-AMP complex. This conclusion is consistent with the crystallographic and structural data available at present.

Although D.discoideum amoebae do not bind AMP at their surface if they are not disrupted, total cell lysates display high levels of AMP binding activity specifically associated with the plasma membrane. The binding of AMP is not competed by adenosine and only poorly by ADP and ATP. The AMP binding sites have a single affinity of 0.6 μM for AMP; the association and dissociation rate constants are respectively 8×10 3 sec −1 M −1 and 4.8 ×10 −3 sec −1 . The AMP binding occurs at a site distinct from the cAMP binding site and from the catalytic site of a membrane bound enzyme.

It is now well established that adenosine 5′-monophosphate (AMP) is a specific, allosteric, noncompetitive inhibitor of fructose 1,6-diphosphatase (FDPase) with cooperative interactions (1). However, our initial studies on the binding of 14C-AMP to rabbit liver FDPase using equilibrium dialysis, gel filtration, and density gradient ultracentrifugation failed to show any detectable binding in the absence of substrate. Further experiments proved that fructose 1,6-diphosphate (FDP) was an essential cooperative factor for AMP binding with the enzyme. This communication presents three lines of evidence in support of this conclusion.

A method is described for the preparation of highly purified fructose 1,6-diphosphatase (EC 3.1.3.11) from the liver of the fish Genypterus chilensis. The purification procedure consists of only three steps, extraction at pH 3.5, ammonium sulfate fractionation, and P-cellulose chromatography. The purified enzyme appears to be homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. As with other fructose 1,6-diphosphatases, the fish liver enzyme shows allosteric AMP inhibition. The inhibition increases with decreasing temperature, and it is also increased by the presence of 150 m m KCl. However, the cooperative interaction among AMP binding sites is independent of temperature and potassium ions. When studied at the same temperature, fructose 1,6-diphosphatase from G. chilensis is much less sensitive to AMP inhibition than rabbit liver fructose 1,6-diphosphatase. However, a comparative study showed that the AMP inhibition curve obtained for the mammalian liver enzyme at 35° falls between the 5 and 15° experimental curves of the fish liver enzyme, in agreement with the physiological temperature range of G. chilensis (7 to 13°). These results are taken as an example of temperature adaptation at the enzymatic level in which a change in enzyme-effector affinity is sufficient to provide an efficient mechanism for the regulation of fructose 1,6-diphosphatase in this poikilotherm.

Twenty-three structural analogues of AMP were tested for their ability to activate and bind phosphorylase b in the presence and absence of substrate or protamine sulfate (or both). Michaelis constants, maximal velocities, and inhibition constants were measured by nucleotide activation of phosphorylase b and compared with the corresponding dissociation and inhibition constants measured by nucleotide quenching of a pyridoxal 5'-phosphate-associated enzyme fluorescence. The excitation maximum was 347 mµ and the emission maximum was 522 mµ. A sigmoidal saturation curve was obtained for AMP quenching of enzyme fluorescence. This, coupled with the ATP inhibition and the glucose 1-phosphate enhancement of AMP quenching of enzyme fluorescence, identified the site of AMP binding for enzyme fluorescence quenching with the AMP binding site for activation of phosphorylase b. Protamine enhanced the binding of some nucleotides to phosphorylase b but apparently affected only the Vmax of others as measured by nucleotide activation of the enzyme. An increase in enzyme fluorescence associated with addition of ATP and a quenching effect by added AMP or glucose-1-P suggested that the observed quenching was the result of the protein conformational state changes associated with the allosteric properties of the enzyme. Nucleotide analogues of AMP exhibited different heterotropic binding properties with phosphorylase b. Binding studies in the presence and absence of substrate revealed that for some nucleotides the affinity was unchanged while others showed less affinity for enzyme in the presence of substrate. Enzyme activation by bound nucleotide was sensitive to the integrity of both the adenine ring and the 5'-phosphate, but the latter appeared to be the most critical structural feature. Both the 5'-phosphate and purine ring were involved in nucleotide binding.

The interaction between phosphorylase b and AMP has been studied calorimetrically. The dependence of the heat of this interaction upon AMP concentration appears to be biphasic. Ultracentrifugal studies show that AMP at 1 mM induces the formation of a tetrameric phosphorylase b which may be partially dissociated at 10 mM AMP. The specific activity of phosphorylase b is sensitive to preincubation in AMP. The dependence of enzyme activity upon AMP concentration of the incubation medium is also biphasic. A lowering of enzyme activity occurs as AMP concentration is raised to 1 mM. Increasing AMP concentration above 1 mM brings about enzyme activation. All the results indicate the existence of distinct AMP sites on phosphorylase b with different affinities.

Inhibition studies with the photoreactive AMP analog, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), demonstrate that this compound is, like AMP, an allosteric inhibitor of pig kidney and muscle fructose-1,6-biphosphateses. Photolysis of a mixture of purified pig kidney fructose-1,6-biphosphate and 8-azido-[14C]AMP results in the loss of enzyme activity and the reagent is incorporated to the protein. The incorporation of reagent linearly correlates with the loss of enzyme activity. Extrapolation to zero activity correlates with the incorporation of 3.7 mol of reagent/mol of enzyme (i.e. 0.9 per subunit). Thus, 8-azido-AMP appears to be a photoaffinity label for the allosteric AMP binding site of fructose-1,6-biphosphatase.

Fructose 1,6-diphosphatase with optimal activity at neutral pH has been purified from pig kidney cortex. The method permits the isolation of an enzyme ("neutral" fructose 1,6-diphosphatase) which maintains its initial ratio of pH 7.5 to pH 9.3 activity during the course of the purification. The same method, after incubation of the extract at 30°, has been used for the purification of altered forms of the enzyme which show a decreased activity at pH 7.5 and no significant changes of activity at pH 9.3. A comparative enzymatic study of the neutral and altered forms of fructose 1,6-diphosphatase showed that at least three properties are significantly changed upon conversion of the native (neutral) to the altered (alkaline) fructose 1,6-diphosphatase. These changes are: (a) a shift in the pH optimum from neutral to alkaline pH values; (b) loss of activation by potassium ions; (c) diminished sensitivity to AMP inhibition and decreased positive cooperative nteraction among AMP binding sites.