The ACT domain: a small molecule binding domain and its role as a common regulatory element.
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Amino Acid Sequence↗
Biomedical subjects
Gregory A Grant
Publications and source records attributed to Gregory A Grant.
Identification of amino acid residues contributing to the mechanism of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase.
L-Serine inhibits the catalytic activity of Escherichia coli D-3-phosphoglycerate dehydrogenase (PGDH) by binding to its regulatory domain. This domain is a member of the ACT domain family of regulatory domains that are modulated by small molecules. A comparison of the phi and psi torsional angle differences between the crystal structures of PGDH solved in the presence and in the absence of L-serine demonstrated a clustering of significant angle deviations in the regulatory domain. A similar clustering was not observed in either of the other two structural domains of PGDH. In addition, significant differences were also observed at the active site and in the Trp-139 loop. To determine if these residues were functionally significant and not just due to other factors such as crystal packing, mutagenic analysis of these residues was performed. Not unexpectedly, this analysis showed that residues that affected the kcat/Km were grouped around the active site and those that affected the serine sensitivity were grouped in the regulatory domain. However, more significantly, residues that affected the cooperativity of inhibition of activity were identified at both locations. These latter residues represent structural elements that participate in both the initial and the ultimate events of the transfer of cooperative behavior from the regulatory domain to the active site. As such, their identification will assist in the elucidation of the pathway of cooperative interaction in this enzyme as well as in the elucidation of the regulatory mechanism of the ACT domain in general.
Allosteric Regulation↗
Vmax regulation through domain and subunit changes. The active form of phosphoglycerate dehydrogenase.
An active conformation of phosphoglycerate dehydrogenase (PGDH) from Escherichia coli has been obtained using X-ray crystallography. The X-ray crystal structure is used to examine the potential intermediates for V(max) regulation, for the redox reaction, and for cooperative effects of serine binding. The crystal structure at 2.2 A resolution contains bound NAD(+) cofactor, either sulfate or phosphate anions, and alpha-ketoglutarate, a nonphysiological substrate. A PGDH subunit is formed from three distinct domains: regulatory (RBD), substrate (SBD), and nucleotide binding (NBD). The crystal conformation of the homotetramer points to the fact that, in the absence of serine, coordinated movement of the RBD-SBD domains occurs relative to the NBD. The result is a conformational change involving the steric relationships of both the domains and the subunits. Within the active site of each subunit is a bound molecule of alpha-ketoglutarate and the coenzyme, NAD. The catalytic or active site cleft is changed slightly although it is still solvent exposed; therefore, the catalytic reaction probably involves additional conformational changes. By comparing the inhibited with the uninhibited complex, it is possible to describe changes in conformation that are involved in the inhibitory signal transduction of serine.
Carbohydrate Dehydrogenases↗
D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes.
D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize alpha-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I(0.5) = 30 microm. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.
Amino Acid Motifs↗
Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: extreme asymmetry in a tetramer of identical subunits.
Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first D-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of D-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 A. This enzyme represents a second structural motif of the D-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of approximately 180 degrees around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits.
Amino Acid Motifs↗
Multiconformational states in phosphoglycerate dehydrogenase.
Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, serine. The regulation occurs through a V(max) mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 A crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated approximately 42 degrees relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V(max) regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.
Allosteric Site↗
Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers.
A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.
Allosteric Regulation↗
Methods for analyzing cooperativity in phosphoglycerate dehydrogenase.
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Carbohydrate Dehydrogenases↗
Pancreatic islets and insulinoma cells express a novel isoform of group VIA phospholipase A2 (iPLA2 beta) that participates in glucose-stimulated insulin secretion and is not produced by alternate splicing of the iPLA2 beta transcript.
Many cells express a group VIA 84 kDa phospholipase A(2) (iPLA(2)beta) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA(2)beta in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA(2)beta in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA(2)beta participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA(2)beta-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA(2)beta protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA(2)beta transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA(2)beta, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA(2)beta tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA(2)beta residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA(2)beta catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA(2)beta activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA(2)beta and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA(2)beta-immunoreactive protein suggests that a signal transduction role of iPLA(2)beta in the native beta-cell might be attributable to a 70 kDa isoform of iPLA(2)beta.
Alternative Splicing↗
Hybrid tetramers reveal elements of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase.
d-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when l-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by approximately 58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.
Allosteric Site↗
Identification of PTH-amino acids by HPLC.
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Amino Acids↗
Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding.
The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.
Allosteric Site↗
De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal.
Escherichia coli 3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibited by serine. Structural studies revealed a homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains: nucleotide, substrate and regulatory. In PGDH, extensive interactions are formed between nucleotide binding domains. A second subunit-subunit interaction occurs between regulatory domains creating an extended beta sheet. The serine-binding sites overlap this interface. In these studies, the nucleotide and substrate domains (NSDs) were subcloned to identify changes in both catalytic and physical properties upon removal of a subunit-subunit interface. The NSDs did not vary significantly from PGDH with respect to kinetic parameters with the exception that serine no longer had an effect on catalysis. Temperature dependent dynamic light scattering (DLS) revealed the NSDs aggregated > 5 degrees C before PGDH, indicating decreased stability. DLS and gel filtration studies showed that the truncated enzyme formed a tetramer. This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated, closely related dimeric enzymes. Expression of the regulatory domain, to study conformational changes induced by serine binding, yielded a product that by CD spectra contained stable secondary structure. DLS and pulsed field gradient NMR studies of the regulatory domain showed the presence of higher oligomers instead of the predicted dimer. We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.
Allosteric Regulation↗