Purification and sequence determination of guanylate kinase from pig brain

Zschocke, P.; Schiltz, Emile; Schulz, Georg E.

doi:10.1111/j.1432-1033.1993.tb17757.x

Cited by 14 publications

(9 citation statements)

References 33 publications

(29 reference statements)

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“…SAP90 contains in its carboxy-terminus (aa 534-724) a region that is very similar to the yeast and the mammalian GKs [10][11][12]. This region of GK in SAP90 share 37 and 39% identical aa to the GK from yeast and from bovine retina, respectively.…”

Section: Gmp Binding Activitymentioning

confidence: 99%

“…(ii) A src homology 3 (SH3) domain, found in a variety of proteins involved in signal transduction processes which are mediated by direct protein-protein interactions (see review [9]). (iii) A carboxy-terminal region similar to the guanylate kinases (GK) from yeast [10] and from mammals [11,12]. GK (ATP:GMP phosphotransferase EC 2.7.4.8) catalyzes the phosphorylation of GMP at the expense of ATP according to the reaction GMP + ATP + Mg > = GDP + ADP + Mg 2÷.…”

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confidence: 99%

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Nucleotide bindind by the synapse associated protein SAP90

Kistner

Garner²,

Linial

1995

FEBS Letters

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The rat synapse associated protein SAP90 is a member of a superfamily of potential guanylate kinases localized at cell-cell contact sites. This superfamily includes the synapse associated protein SAP97, a close relative of SAP90, the Drosophila tumor suppressor gene product dlg-Ap, the mammalian zonula occludens proteins ZO-1 and ZO-2 and the erythrocyte protein p55. Here we show that SAP90 specifically binds GMP in the micromolar range while binding to ATP, GDP and ADP is at a much lower affinity (10-25 mM), whether or not binding is detected for other guanine and adenine nucleotides. No guanylate kinase activity of SAP90 was detected under our experimental conditions. The importance of the GMP binding capacity per se and an evolutionary role for conserving of the guanylate kinase domain in this superfamily are discussed.

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Section: Gmp Binding Activitymentioning

confidence: 99%

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confidence: 99%

Nucleotide bindind by the synapse associated protein SAP90

Kistner

Garner²,

Linial

1995

FEBS Letters

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“…The amino acid sequence of porcine brain GK was determined by peptide sequencing (5). Yeast GK shares 44.8% identity with E. coli GK, 51% identity with porcine GK, and 55% identity with bovine GK.…”

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confidence: 99%

Kinetic and Thermodynamic Characterizations of Yeast Guanylate Kinase

Zhang

Yan

1996

Journal of Biological Chemistry

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Yeast guanylate kinase was expressed at high level in Escherichia coli using pET-17b vector. It was purified to homogeneity by a simple two-column procedure with an average yield of ϳ100 mg/liter. The steady-state kinetic parameters for both forward and reverse reactions were determined by initial velocity measurements. The turnover numbers (k cat ) were 394 s ؊1 for the forward reaction (formation of ADP and GDP) and 90 s ؊1 for the reverse reaction (formation of ATP and GMP). K m values were 0.20, 0.091, 0.017, and 0.097 mM for MgATP, GMP, MgADP, and GDP, respectively. Analysis of the initial velocity patterns indicated a sequential mechanism. GMP was found to have partial substrate inhibition. The substrate inhibition was not competitive with MgATP and could be attributed to formation of the abortive complex guanylate kinase⅐MgADP⅐GMP. The equilibrium constant of the reaction was measured under various conditions by NMR and a radiometric assay. The results showed that the steady-state kinetic parameters were consistent with the thermodynamic constant. NMR titration and equilibrium dialysis showed that both substrates and products could bind to free guanylate kinase. The dissociation constants were 0.090, 0.18, 0.029, 0.084, and 0.12 mM for MgATP, ATP, GMP, MgADP, and GDP, respectively. Viscosity-dependent kinetics was used to identify the rate-limiting steps of the reaction. The results indicated that the reaction rate is largely controlled by the chemical step. Guanylate kinase (GK)1 catalyzes the reversible phosphoryl transfer from ATP to GMP in the presence of Mg 2ϩ (1). The enzyme is essential for converting GMP to GDP and therefore synthesis of GTP. It plays an important role in the cGMP cycle (2) and is also required for metabolic activation of the antiviral drugs acyclovir and gancyclovir (3, 4).GK has been purified to various degrees of purity from several sources (5 and references therein), but detailed characterization has been hampered by its low abundance. Mammalian GK has been characterized by steady-state kinetics (1, 2), but no further mechanistic studies have been reported. It was not until 1989 that yeast GK was purified to homogeneity, and its amino acid sequence was determined by peptide sequencing (6). The crystal structure of yeast GK in complex with GMP was solved and refined shortly afterwards at 2-Å resolution (7, 8).The genes for yeast GK, Escherichia coli GK, and bovine GK were recently cloned (9 -11). The amino acid sequence of porcine brain GK was determined by peptide sequencing (5). Yeast GK shares 44.8% identity with E. coli GK, 51% identity with porcine GK, and 55% identity with bovine GK. It is noteworthy that, unlike the yeast and mammalian GKs, which are monomeric, E. coli GK is tetrameric at low salt conditions and dimeric at high salt conditions (10). Interestingly, several proteins, including the protein encoded by Drosophila tumor suppresser gene dlg-A (12), a rat presynaptic density protein (SAP90) (13), a rat postsynaptic density protein (PSD-95) (14), and the major pa...

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“…As with other enzymes involved in nucleotide metabolism, guanylate kinase is a target for cancer chemotherapy and is inhibited by the potent antitumor drug, 6-thioguanine (3)(4)(5). Guanylate kinase activity is also required for the potentiation of antiviral drug activity in virus-infected cells.…”

mentioning

confidence: 99%

Cloning, Characterization, and Modeling of Mouse and Human Guanylate Kinases

Brady

Kokoris

Fitzgibbon

et al. 1996

Journal of Biological Chemistry

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Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways. Despite its involvement in antiviral drug activation in humans and in mouse model systems and as a target for chemotherapy, the human and mouse primary structures have never been elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinase were isolated from mouse B-cell lymphoma and human peripheral blood lymphocyte cDNA libraries. Multiple tissue Northern blots demonstrated an mRNA species of approximately 1 kilobase for both mice and humans in all tissue types examined. The mouse cDNA is predicted to encode a 198-amino acid protein with a molecular mass of 21,904 daltons. The human cDNA is predicted to encode a 197-amino acid protein with a molecular mass of 21,696 daltons. These proteins share 88% sequence identity with each other and 52-54% identity with the yeast guanylate kinase. Molecular modeling using the yeast diffraction coordinates indicates a high degree of conservation within the active site and maintenance of the overall structural integrity, despite a lack of similarity along the periphery of the enzyme.Guanylate kinase (GMK, 1 ATP:GMP phosphotransferase; EC 2.7.4.8) catalyzes the reaction (d)GMP ϩ ATP 3 (d)GDP ϩ ADP where (d)GMP indicates GMP or dGMP. In addition to being a critical enzyme in the biosynthesis of GTP and dGTP, guanylate kinase functions in the recovery of cGMP (cGMP 3 GMP 3 GDP 3 GTP 3 cGMP) and is, therefore, thought to regulate the supply of guanine nucleotides to signal transduction pathway components (1, 2).As with other enzymes involved in nucleotide metabolism, guanylate kinase is a target for cancer chemotherapy and is inhibited by the potent antitumor drug, 6-thioguanine (3-5). Guanylate kinase activity is also required for the potentiation of antiviral drug activity in virus-infected cells. Activation of the anti-herpes guanosine nucleoside analogs, acyclovir and ganciclovir, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase (6, 7).Although guanylate kinase is a key enzyme for cancer chemotherapy and antiviral drug activation in humans, the gene encoding this important enzyme has not been described. In this study, we describe the isolation, expression, and functional analysis of both human and mouse guanylate kinases in an effort to aid improvements in antiviral and tumor chemotherapeutic approaches through molecular modeling and rational drug design. EXPERIMENTAL PROCEDURESMaterials-Restriction enzymes, deoxynucleoside triphosphates, and random-primed DNA labeling kits were purchased from Boehringer Mannheim. Reagents for enzyme assays were purchased from Sigma.[␣-32 P]dCTP (3000 Ci/mmol) and 14 C-labeled protein molecular weight markers were purchased from Amersham Corp. 35S Express protein labeling mix was purchased from DuPont NEN. Biotinylated thrombin, pET23d vector, and strepavidin beads were obtained from Novagen (Madison, WI). Multiple tissue ...

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Purification and sequence determination of guanylate kinase from pig brain

Cited by 14 publications

References 33 publications

Nucleotide bindind by the synapse associated protein SAP90

Nucleotide bindind by the synapse associated protein SAP90

Kinetic and Thermodynamic Characterizations of Yeast Guanylate Kinase

Cloning, Characterization, and Modeling of Mouse and Human Guanylate Kinases

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