Kinetic and Thermodynamic Characterizations of Yeast Guanylate Kinase

MembraneTissue development, differentiation, and physiology require specialized cellular adhesion and signal transduction at sites of cell-cell contact. Scaffolding proteins that tether adhesion molecules, receptors, and intracellular signaling enzymes organize macromolecular protein complexes at cellular junctions to integrate these functions. One family of such scaffolding proteins is the large group of membrane associated guanylate kinases (MAGUKs) 1 (1-4). Genetic studies have highlighted the critical roles for MAGUK proteins in the development and physiology of numerous tissues from a variety of metazoan organisms. Mutation of Drosophila discs large (dlg) disrupts epithelial septate junctions and causes overgrowth of the imaginal discs (5). Similarly, mutation of lin-2, a related MAGUK in Caenorhabditits elegans, blocks vulval development (2), and mutation of the postsynaptic density protein PSD-95 impairs synaptic plasticity in mammalian brain (6). MAGUK proteins are composed of a set of protein-protein interaction domains that explain their important roles in organizing protein complexes at sites of cell-cell contact. MAGUKs contain L27 heterodimerization domains, one or three PDZ domains, an SH3 domain and a C-terminal domain homologous to guanylate kinase (GK) (7,8). PDZ domains are small modular motifs that contain a single peptide binding groove that associates with specific sequences often found at the extreme C termini of interacting ion channels (9, 10), cell adhesion molecules (11, 12), and other membrane-associated proteins (13-16). Multiple PDZ domains in PSD-95 organize and accelerate signal transduction at synapses by linking receptors to downstream signaling enzymes (17,18).Whereas the functional roles for PDZ domains in MAGUKs are well established, functions for the SH3 and GK domains are less certain. SH3 domains classically bind to proline-rich motifs (19 -23); however, the structure of the PSD-95 SH3 domain suggests that such interactions are unlikely because a conserved helix in MAGUK SH3 domains occludes the canonical polyproline binding site (24,25). A variety of high affinity protein ligands have been identified for GK domains from several MAGUKs (26 -30), but it is not yet clear how these interactions regulate MAGUK functions. In addition to binding exogenous ligands, protein fragments containing the proposed SH3 and GK regions of MAGUK proteins interact with each other (31-34). The crystal structure of the SH3GK region of PSD-95 reveals that this interaction is the assembly of the SH3 fold from discontinuous structural components (24,25). This SH3/GK interaction, may oligomerize MAGUK scaffolds, but factors that regulate intermolecular SH3 assembly remain uncertain. Despite our limited understanding of the biochemical roles for the SH3 and GK domains, these regions are clearly critical as most genetically identified mutations of MAGUKs occur in the SH3 and GK domains (2, 5).The GK domains of MAGUKs share 40% sequence homology with authentic GK enzymes, which phosphorylate GMP to for...

Section: Resultssupporting

confidence: 74%

Functional Analysis of the Nucleotide Binding Domain of Membrane-associated Guanylate Kinases

Olsen

Bredt²

2003

“…In all cases, GTP reversed inhibition by excess UMP. Because inhibition by excess nucleoside monophosphate has also been observed with other NMP kinases such as E. coli adenylate kinase (25,26) and CMP kinase (27) and yeast GMP kinase (28), several common causes might be invoked to explain this phenomenon. Binding of UMP to the MgATP site is excluded, as inhibition is not competitive with MgATP.…”

Section: Discussionmentioning

confidence: 99%

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

Evrin

Străuţ

Slavova‐Azmanova

et al. 2007

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K m for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Grampositive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T % R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.Bacterial UMP kinases represent a particular subfamily of NMP 2 kinases (1, 2). They do not share any significant sequence homology with other known NMP kinases and exist in solution as stable hexamers. A first structural model of Escherichia coli UMP kinase (3) based on the conservation of the carbamate kinase and N-acetylglutamate kinase folds (4, 5) helped to better rationalize previous site-directed mutagenesis experiments (6). The crystal structure of E. coli UMP kinase (7) indicated a similar fold between its monomers and N-acetylglutamate kinase, a dimeric enzyme (4, 5). However, the quaternary structure assembly of these two proteins is completely different (7). Deposited crystal structures of UMP kinases from other bacteria such as Pyrococcus furiosus (8), Neisseria meningitidis (Protein Data Bank code 1YBD), Hemophilus influenzae (code 2AIF), and Streptococcus pyogenes (code 1Z9D) show threedimensional structures very similar to that of the E. coli enzyme. The residues essential for binding nucleotide substrates and catalysis are conserved among all bacterial UMP kinases ( Fig. 1) (3, 9). Consequently, the active sites of these enzymes and the phosphoryl transfer mechanisms are most probably similar.Comparison of the biochemical properties of recombinant UMP kinases from Gram-negative E. coli (1, 2) and Gram-positive Streptococcus pneumoniae (10) indicated significant differences in their kinetic properties particularly in their regulation by nucleotides. Unlike the E. coli enzyme, U...

“…Notwithstanding these limitations, we found that at low concentrations (2 M GDP, 10 M GMPK), no complex with GTP was formed within the first 10 min and complexes of GMPK⅐GDP (22,448) were almost exclusively detected. The formation of GMPK⅐GTP (22,528) correlated with the formation of GMPK⅐GDP 2 (22,891).…”

Section: Resultsmentioning

confidence: 99%

“…All these findings can be explained with a simple modification of the commonly employed reaction scheme (22).…”

Section: Resultsmentioning

confidence: 99%

Binding of Nucleotides to Guanylate Kinase, p21 , and Nucleoside-diphosphate Kinase Studied by Nano-electrospray Mass Spectrometry

Prinz

Lavie

Scheidig

et al. 1999

The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4.8), the transfer of HPO 3 between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg 2؉ binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P 1 -(5-guanosyl)-P 5 -(5-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21 ras and GDP. Nucleosidediphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO 4 2؊ ) with presumably high affinity and slow exchange rate. The binding of phosphate (PO 4 3؊ ) could be inferred indirectly from competition with SO 4 2؊ .Nano-electrospray mass spectrometry (1) is a simple modification of electrospray mass spectrometry, in which the diameter of the nanospray capillary is reduced to a few micrometers. The method is relatively tolerant toward salts because liquid droplets formed by nanospray off small capillaries are smaller than droplets formed by standard pneumatically assisted electrospray methods (2). Smaller droplets lead to less total amount of salt/droplet and, at protein concentrations of approximately 1 molecule/droplet, to less salt on the protein after evaporation of the droplet. Therefore, this method has been successfully applied to the study of proteins and peptides where salt adducts are a common obstacle. It has been shown that complexes formed between ligands and proteins could be detected by means of electrospray mass spectrometry (3-7). We have employed nano-electrospray mass spectrometry to test its suitability for systematic studies of nucleotide binding to proteins in aqueous solutions.Three different nucleotide-binding proteins were studied: 1) guanylate kinase (GMPK) 1 (EC 2.7.4.8), which belongs to a class of enzymes that catalyze the ATP-dependent phosphorylation of monophosphonucleotides into diphosphonucleotides. The crystal structure of GMPK from Saccharomyces cerevisiae complexed with its substrate GMP is known (8). 2) p21 ras , which belongs to a well investigated class of small GTP-binding proteins that are essential for the regulation of a wide variety of cellular processes (9). Its structure had been determined in the presence of a GTP analogue (10). 3) nucleoside-diphosphate kinase (NDK) (EC 2.7.4.6), which exhibits a much lower substrate specificity than the above enzymes. It is involved in the synthesis of all nucleoside triphosphates other th...