Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The functionally diverse actin, hexokinase, and hsp70 protein families have in common an ATPase domain of known three-dimensional structure. Optimal superposition of the three structures and alignment of many sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are involved in ATP binding and in a putative interdomain hinge. From the multiple sequence alignment in these motifs a pattern of amino acid properties required at each position is defined. The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method of generalizing from observed amino acid frequencies to amino acid fitness at each sequence position. A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA. These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and IIa of hexokinase, actin, and Hsc70, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying functional state changes. A common evolutionary origin for all of the proteins in this class is proposed.
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PMID:An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. 132 28

A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. The Michaelis-Menten constants of the two substrates with the enzyme are 70 microM for the nucleoside and 26 microM for P(i); the kcat is 40 s-1 (25 degrees C). The assay was shown to quantitate P(i) in solution at concentrations at least down to 2 microM. It can be used to measure the kinetics of P(i) release from phosphatases, such as GTPases and ATPases, by coupling the two enzymic reactions. The utility of this assay was shown by three test systems: glycerol kinase plus D-glyceraldehyde acting as an ATPase and actin-activated myosin ATPase, and myosin subfragment 1, hydrolyzing a single turnover of ATP, releasing P(i) with a rate constant the same as the steady-state ATPase activity.
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PMID:A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. 153 9

Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.
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PMID:Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. 182 89

Hexachlorocyclohexanes (HCCH) are chlorinated analogs of inositol; the alpha, beta, gamma, and delta isomers of HCCH have the stereochemical configurations of (+/-)-, scyllo-, muco-, and myo-inositol, respectively. To assess their potential as specific tools for the study of agonist-stimulated phosphoinositide metabolism, we examined the effects of these four HCCH isomers on phosphatidylinositol (PI) synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase), PI:inositol exchange enzyme, and several membrane-associated enzymes unrelated to inositol metabolism. In pancreas microsomes, in the presence of saturating myo-inositol, the alpha, beta, gamma, and delta isomers (4 mM) inhibited PI synthase activity by 9, 4, 22, and 69%, respectively. Half-maximal inhibition by delta-HCCH occurred at 0.25 mM. A similar pattern of HCCH inhibition was obtained using n-octylglucopyranoside-solubilized and partially purified PI synthase preparations. The inhibition by delta-HCCH was noncompetitive versus myo-inositol. The PI:inositol exchange enzyme in mouse pancreas microsomes was inhibited 90% by 1 mM delta-HCCH in the presence of 0.25% Triton X-100, but not in its absence; half-maximal inhibition occurred with 0.5 mM delta-HCCH. delta-HCCH (4 mM) also inhibited to varying extents the following enzymes: pancreas CDP-choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (75%), brain and erythrocyte (Na+,K+)-ATPase (87 and 70%), brain and erythrocyte Mg2+-ATPase (38 and -5%), brain 1,2-diacyl-sn-glycerol kinase (22%), and liver glucose 6-phosphatase (16%). gamma-HCCH (4 mM) inhibited these enzymes to a lesser extent, or not at all. The order of inhibition by HCCH stereoisomers was the same as the order of their saturation level in phospholipid vesicles (delta greater than gamma greater than alpha greater than beta). This suggests that the inhibitory action is due to insertion of the compounds either into hydrophobic domains of the enzymes or into annular lipid. The results indicate that the HCCHs are not selective inhibitors of inositol metabolism.
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PMID:Inhibition of phosphatidylinositol synthase and other membrane-associated enzymes by stereoisomers of hexachlorocyclohexane. 257 70

The energy requirement for translocation of alkaline phosphatase and the outer membrane protein OmpA into Escherichia coli membrane vesicles was studied under conditions that permit posttranslational translocation and, hence, prior removal of various components necessary for protein synthesis. Translocation could be supported by an ATP-generating system or, less well, by the protonmotive force generated by D-lactate oxidation; the latter might act by generating ATP from residual bound nucleotides. However, when protonmotive force inhibitors were used or when ATP was further depleted by E. coli glycerol kinase, D-lactate no longer supported the translocation. Furthermore, ATP could still support protein translocation in the presence of proton uncouplers or with membranes defective in the F1 fraction of the H+-ATPase. We conclude that ATP is required for protein translocation in this posttranslational system (and probably also in cotranslational translocation); the protonmotive force may contribute but does not appear to be essential.
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PMID:ATP is essential for protein translocation into Escherichia coli membrane vesicles. 286 5

A method has been developed for calculating rate constants for dehydration of aldehydes that induce ATPase reactions by kinases and where 18O is transferred from the aldehyde or its hydrate to inorganic phosphate during the reaction. The method involves measurement of the fraction of 18O in phosphate by 31P NMR after the ATPase reaction has proceeded for several minutes with zero-order kinetics. The reaction is started by addition of the aldehyde in a small volume of H2 18O, and the speed of washout of 18O by reversible dehydration relative to the rate of the ATPase reaction allows calculation of the rate constants if the hydration equilibrium constant is known from the proton NMR spectrum of the aldehyde. Dehydration rate constants (s-1 at pH 8-8.5, 0.1 M buffer, 25 degrees C) for the following aldehydes (all over 95% hydrated) and kinases used are as follows: D-glyceraldehyde with glycerokinase, 0.03; 2,5-anhydro-D-mannose 6-phosphate with fructose-6-phosphate kinase, 0.025; 2,5-anhydro-D-mannose or 2,5-anhydro-D-talose with fructokinase, 0.029 and 0.017, respectively; D-gluco-hexodialdose with hexokinase, 0.068. With betaine aldehyde and choline kinase or glyoxylate and pyruvate kinase, no 18O was transferred to phosphate during the ATPase reactions. However, the dehydration rate constant for glyoxylate (0.007 s-1 at pH 7 extrapolated to zero buffer concentration and up to 0.11 s-1 at pH 9.0 with 0.3 M buffer) was determined by extrapolating the initial rate of reduction of the free aldehyde catalyzed by lactate dehydrogenase to infinite enzyme levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for determining rate constants for dehydration of aldehyde hydrates. 609 90

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the presence of keto or aldehyde analogues (N-methylhydantoic acid, D-glyceraldehyde 3-phosphate, and acetaldehyde, respectively), possibly because of the absence of an acid-base catalytic group in the latter two cases. These analogues were competitive inhibitors vs. the normal substrates, and in the latter case, the hydrate of acetaldehyde was shown to be the inhibitory species on the basis of the deuterium isotope effect on the inhibition constant.
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PMID:Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases. 609 91

Procyclic culture forms of Trypanosoma brucei stock 427 have been screened for the presence of enzymes involved in glycolysis, mitochondrial energy metabolism and threonine degradation. The enzyme activities in the procyclics were compared with those of the blood stream forms. The specific activities of glycolytic enzymes represented 30-70% of the respective levels in the blood stream form, except for hexokinase which was 25-fold reduced. Cell fractionation showed that the enzymes involved in the early sequence of the glycolytic pathway, i.e. from hexokinase to phosphoglycerate kinase, and the enzymes NAD+-linked glycerol-3-phosphate dehydrogenase and glycerol kinase were all present in glycosomes equilibrating at a density of 1.23 g/cm3 in sucrose gradients. Malate dehydrogenase was 8-fold more active in procyclics than in bloodstream forms. This increase in activity was the result of the appearance of malate dehydrogenase in the glycosomes of the procyclics, in addition to mitochondrial and cell-sap activities which were present in both stages of the life cycle. Glycosomes contained part of the adenylate kinase activity, which was also associated with the mitochondrion. Succinate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase, together with oligomycin-sensitive ATPase, were located in the mitochondrion which had a density in sucrose ranging from 1.16 to 1.18 g/cm3. This organelle also contained L-threonine 3-dehydrogenase and carnitine acetyltransferase, two enzymes involved in threonine catabolism. The latter two enzymes had activities which were, respectively, 15-and 13-fold higher in the procyclics than in the bloodstream form. Mitochondrial sn-glycerol-3-phosphate dehydrogenase was decreased 4-fold.
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PMID:Localization of malate dehydrogenase, adenylate kinase and glycolytic enzymes in glycosomes and the threonine pathway in the mitochondrion of cultured procyclic trypomastigotes of Trypanosoma brucei. 680 9

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.
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PMID:Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion. 1009 Jul 37

Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is inhibited allosterically by phosphotransferase system protein IIA(Glc). The primary structures of these related kinases contain 501 amino acids, differing at 117. IIA(Glc) inhibition is transplanted from E. coli glycerol kinase into H. influenzae glycerol kinase by interconverting only 11 of the differences: 8 residues that interact with IIA(Glc) at the allosteric binding site and 3 residues in the conserved ATPase catalytic core that do not interact with IIA(Glc) but the solvent accessible surface of which decreases when it binds. The three core residues are crucial for coupling the allosteric site to the conserved catalytic core of the enzyme. The site of the coupling residues identifies a regulatory locus in the sugar kinase/heat shock protein 70/actin superfamily and suggests relations between allosteric regulation and the active site closure that characterizes the family. The location of the coupling residues provides empirical validation of a computational model that predicts a coupling pathway between the IIA(Glc)-binding site and the active site [Luque, I. & Freire, E. (2000) Proteins Struct. Funct. Genet. Suppl. 4, 63-71]. The requirement for changes in core residues to couple the allosteric and active sites and switching from inhibition to activation by a single amino acid change are consistent with a postulated mechanism for molecular evolution of allosteric regulation.
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PMID:Transplanting allosteric control of enzyme activity by protein-protein interactions: coupling a regulatory site to the conserved catalytic core. 1216 59


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