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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.
Mol Biochem Parasitol 1992 Sep
PMID:Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias. 143 59

The structure of adenylate kinase from Escherichia coli ligated with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate has been determined by X-ray diffraction and refined to a resolution of 1.9 A. The asymmetric unit of the crystals contains two copies of the complex, the structures of which agree well with each other. One of these copies is less well ordered in the crystals than the other, it shows generally higher temperature factors. The molecular packing in the crystals is discussed and correlated to crystal habit and anisotropic X-ray diffraction. The bound inhibitor simulates well the binding of substrates ATP and AMP, which are clearly assigned. The alpha-phosphate of AMP is well positioned for a nucleophilic attack on the gamma-phosphate of ATP. The observed structure readily allows the construction of a stabilized pentaco-ordinated transition state, as proposed for the known in-line mechanism of the enzyme, with nucleophile and leaving group in the apical positions of a trigonal bipyramid. The kinetic data of numerous mutations reported in the literature are correlated with the detailed structure of the enzyme. The mutants were classified. The concomitant increase of the Michaelis constants for ATP and AMP in the group of mutants that modify only the ATP-binding site cannot be explained.
J Mol Biol 1992 Mar 05
PMID:Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. 154 97

Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeast is targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disrupted yeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.
Mol Gen Genet 1992 Jun
PMID:A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria. 162 94

The aim of this study was to determine whether acute changes in [Mg2+]free occur during increased hydrolysis of cytosolic ATP, and whether these changes were of sufficient magnitude to be involved in the modulation of myocardial metabolism. 31P-NMR was used to estimate free cytosolic Mg2+ levels ([Mg2+]free) during hypoxia, isoproterenol stimulation, and graded low-flow ischemia in crystalloid perfused, isovolumic rat hearts. Cytosolic [Mg2+]free was calculated to be 0.73 +/- 0.12 mM in control hearts (100 mmHg hydrostatic pressure, 95% O2, n = 18). Cytosolic [Mg2+]free increased gradually during 10 min periods of hypoxia (65%, 50%, 35%, 5% O2), and 20 min infusions of isoproterenol (0.4, 3.0, 75 nM), to maximum values greater than 250% of control (P less than 0.05). During 8 min periods of graded low-flow ischemia (12.0, 7.2, 5.3, 3.4, and 1.6 ml/min/g), [Mg2+]free did not change significantly. [Mg2+]free displayed an inverse linear correlation with total cytosolic [ATP] during isoproterenol infusion (r = 0.87), and an exponential correlation during hypoxia (r = 0.82). The data indicate that acute changes in cytosolic [Mg2+]free can occur during conditions of net ATP hydrolysis although changes in ATP alone do not appear to be solely responsible for the changes in [Mg2+]free. Since the magnitude of the changes in [Mg2+]free are sufficient to alter equilibria of enzymes such as creatine kinase and myokinase, it is possible that these changes are involved in the acute modulation of myocardial metabolism.
J Mol Cell Cardiol 1991 Sep
PMID:Cytosolic free magnesium in stimulated, hypoxic, and underperfused rat heart. 165 49

Irradiation of adenylate kinase (AK) from chicken muscle with 300-400-nm light in the presence of 0.25 mM vanadate ion first inactivated the enzyme and then cleaved the polypeptide chain near the NH2 terminus. The addition of the multisubstrate analogue, P1,P5-bis(5'-adenosyl) pentaphosphate, prevented both effects. ATP, but not AMP, blocked both inactivation and cleavage in a saturable manner, suggesting that both effects were due to modification at the ATP-binding site. The polypeptide products of the photocleavage were isolated by HPLC and characterized by amino acid composition, peptide sequencing, and mass spectral analyses. The predominant (greater than 90%) small peptide fragment contained the first 16 amino acids from the amino terminus of the enzyme. The amino terminus of this peptide contained an acetylated serine, and the "carboxy" terminus was modified by a cyclized gamma-aminobutyric acid which originated from photooxidation and decarboxylation of proline-17 by vanadate. Edman sequencing indicated that the majority of the large peptide fragment (Mr approximately 19,500) was amino-terminal blocked, but a small portion was sequenceable starting at either glycine-18 (7%) or serine-19 (2%). These studies indicate that in the ATP-AK complex proline-17 is close to the phosphate chain of ATP but not AMP, consistent with the latest evaluation of nucleotide-binding sites on mitochondrial matrix AK by X-ray crystallography [Diederichs, K., & Schulz, G.E. (1991) J. Mol. Biol. 217, 541-549]. Furthermore, this is the first report that an amino acid other than serine can be involved in vanadate-promoted photocleavage reactions.
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PMID:Vanadate catalyzes photocleavage of adenylate kinase at proline-17 in the phosphate-binding loop. 173 8

The role of one of the histidine residues present in many adenylate kinases (H36 in the porcine cytosolic enzyme) is highly disputed. We thus studied the yeast enzyme (AKye) containing this His residue. AKye is highly homologous to the Escherichia coli enzyme (AKec), a protein that is already well characterized by NMR [Vetter et al. (1990) Biochemistry 29, 7459-7467] and does not contain the His residue in question. In addition, discrepancies between solution structural and X-ray crystallographic studies on the location of the nucleotide binding sites of adenylate kinases are clarified. One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to investigate AKye and its complex with the bisubstrate analogue P1,P5-bis(5'-adenosyl)pentaphosphate (AP5A). The well-resolved spectra of AKye allowed identification of nearly all detectable resonances originating from aromatic side chain protons (12 out of 15 spin systems). From these studies, all aromatic residues of AKec involved in the binding of ATP.Mg2+ have functional analogues in AKye. The AMP site seems to make no contacts to aromatic side chains, neither in the AKye.AP5A.Mg2+ nor in the AKec.AP5A.Mg2+ complexes, so that it is presently not possible to localize this binding site by NMR. The ATP site of AKye is located near residues W210 and H143 in a position similar to the ATP site of the E. coli enzyme. In combination with the recent X-ray results on the AP5A complexes AKye and AKec and the GMP complex of guanylate kinase [Stehle, T., & Schultz, G. E. (1990) J. Mol. Biol. 221, 255-269], the latter one leading to the definition of the monophosphate site, the problem of the location of the nucleotide sites can be considered to be solved in a way contradicting earlier work [for a review, see Mildvan, A. S. (1989) FASEB J. 3, 1705-1714] and denying the His residue homologous to H36 in porcine adenylate kinase a direct role in substrate binding.
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PMID:Complexes of yeast adenylate kinase and nucleotides investigated by 1H NMR. 185 Jun 18

Many proteins that bind purine nucleotide triphosphates have a type A sequence motif. Only two classes of structures for such proteins are so far available from X-ray crystallography. We examined the tertiary structures of representatives of the two classes, porcine cytoplasmic adenylate kinase and Escherichia coli translational elongation factor Tu. Comparison of the two proteins suggests that the A motif may be just one part of a larger common core structure consisting of four parallel strands of beta-sheet sandwiched between four alpha-helices. This compact core structure comprises over one half of each protein. We speculate that A motif proteins have diverged from a common ancestor having this core structure.
J Mol Biol 1991 Oct 05
PMID:Evidence for an ancestral core structure in nucleotide-binding proteins with the type A motif. 194 27

A chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. lic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. ORF2 encodes the UDP-galactose-4-epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. ORF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3' end of ORF1, all of galE, and the 5' end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.
Mol Microbiol 1991 May
PMID:Molecular analysis of a complex locus from Haemophilus influenzae involved in phase-variable lipopolysaccharide biosynthesis. 195 82

The crystal structure of the complex between adenylate kinase from bovine mitochondrial matrix and its substrate AMP has been refined at 1.85 A resolution (1 A = 0.1 nm). Based on 42,519 independent reflections of better than 10 A resolution, a final R-factor of 18.9% was obtained with a model obeying standard geometry within 0.016 A in bond lengths and 3.2 degrees in bond angles. There are two enzyme: substrate complexes in the asymmetric unit, each consisting of 226 amino acid residues, one AMP and one sulfate ion. A superposition of the two full-length polypeptides revealed deviations that can be described as small relative movements of three domains. Best superpositions of individual domains yielded a residual overall root-mean-square deviation of 0.3 A for the backbone atoms and 0.5 A for the sidechains. The final model contains 381 solvent molecules in the asymmetric unit, 2 x 72 = 144 of which occupy corresponding positions in both complexes.
J Mol Biol 1991 Feb 05
PMID:The refined structure of the complex between adenylate kinase from beef heart mitochondrial matrix and its substrate AMP at 1.85 A resolution. 199 37

Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR. 203 23


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