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

Abstract

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...

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The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

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Novel Genes Coding for Lithotrophic Sulfur Oxidation of Paracoccus pantotrophus GB17

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