Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits.     

Generation,characterization, and heterologous expression of wild-type and up-regulated forms of Arabidopsis thaliana leaf ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch biosynthesis of higher plants, consists of a pair of regulatory large (LS) and catalytically small (SS) subunits. In plants, these subunits are coded by multiple genes resulting in the formation of tissue-specific enzyme forms, which are differentially regulated during plant growth and development. Some AGPase isoforms differ in catalytic and regulatory properties as well as intracellular location. In an effort to gain a better understanding of the role of the leaf AGPase in carbon partitioning and its effect on plant productivity, the Arabidopsis leaf AGPase containing the mature forms of the SS and LS was expressed in a heterologous expression system and characterized enzymatically. The Arabidopsis recombinant AGPase had kinetic values for 3-phosphoglyceric acid, glucose-1-phosphate and Mg(2+) similar to those of the native enzyme. As the N-terminus of the LS has been suggested to be involved in enzyme function, the length of the N-terminal region was extended or shortened. Of the five modified LSs analyzed, only the T5 form lacking six residues of the mature N-terminus was able to form detectable levels of enzyme activity, indicating that the N-terminal region is critical for enzyme function. Two up-regulatory LS mutations that allosterically activate the potato enzyme, a stem isoform, were introduced into the corresponding Arabidopsis LS sequences and co-expressed with wild-type SS. Both modified enzymes showed up-regulatory properties, indicating that these specific residue changes were also operational in the leaf isoform.     

Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.     

Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography

In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.     

Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.     

Insight into the 3D structure of ADP-glucose pyrophosphorylase from rice (Oryza sativa L.)

ADP-glucose pyrophosphorylase (E.C. 2.7.7.27; AGPase) is a key regulatory enzyme that catalyzes the rate-limiting step of starch biosynthesis in higher plants. AGPase consists of pair of small (SS) and large (LS) subunits thereby constituting a heterotetrameric structure. No crystal structure of the native heterotetrameric enzyme is available for any species, thus limiting the complete understanding of structure–function relationships of this enzyme. In this study, an attempt was made to deduce the heterotetrameric assembly of AGPase in rice. Homology modeling of the three-dimensional structure of the LS and SS was performed using the Swiss Model Server, and the models were evaluated and docked using GRAMM-X to obtain the stable heterodimer orientation (LS as receptor and SS as ligand) and then the heterotetrameric orientation. The initial heterotetrameric orientation was further refined using the RosettaDock Server. MD simulation of the representative heterodimer/tetramer was performed using NAMD, which indicated that the tail-to-tail interaction of LS and SS was more stable than the head-to-head orientation, and the heterotetramer energy was also minimized to ?767,011 kcal mol^?1. Subunit–subunit interaction studies were then carried out using the programs NACCESS and Dimplot. A total of 57 interface residues were listed in SS and 63 in LS. The residues plotted by Dimplot were similar to those listed by NACCESS. Multiple sequence alignment of the sequences of LS and SS from potato, maize and rice validated the interactions inferred in the study. RMSD of 1.093 Å was obtained on superimposition of the deduced heterotetramer on the template homo-tetramer (1YP2), showing the similarity between the two structures.     

Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form

The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.     

Investigation of subunit function in ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme. The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.     

Insights into subunit interactions in the heterotetrameric structure of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies.     

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.     

马铃薯AGPase大小亚基功能研究

马铃薯 1,6 二磷酸腺苷葡萄糖焦磷酸化酶 (AGPase)是淀粉合成的限速酶 ,该酶有大、小两个亚基形成异源四聚体。总结了迄今为止已克隆的马铃薯AGPase大、小亚基编码基因、小亚基和底物结合位点的识别、以及大亚基异构调控因子结合位点识别的研究结果 ,提出了大小亚基非自然重组是深入研究AGPase的途径 ,建立体内条件下高效可靠代谢调控研究手段是AGPase研究所必需的。     

Comparative computational analysis of ADP Glucose Pyrophosphorylase in plants

ADP-glucose pyrophosphorylase (AGPase), a key enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Ample evidence has shown that the AGPase catalyzes the rate limiting step in starch biosynthesis in higher plants. In this study, we compiled detailed comparative information about ADP glucose pyrophosphorylase in selected plants by analyzing their structural features e.g. amino acid content, physico-chemical properties, secondary structural features and phylogenetic classification. Functional analysis of these proteins includes identification of important 10 to 20 amino acids long motifs arise because specific residues and regions proved to be important for the biological function of a group of proteins, which are conserved in both structure and sequence during evolution. Phylogenetic analysis depicts two main clusters. Cluster I encompasses large subunits (LS) while cluster II contains small subunits (SS).     

Both subunits of ADP-glucose pyrophosphorylase are regulatory

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.     

The enzyme activity allosteric regulation model based on the composite nature of catalytic and regulatory sites concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

Abstract

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure.

The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric

The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.     

AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.     

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.