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:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]
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PMID:Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site. 851 1

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldolase, by binding these cytoplasmic enzymes at its N-terminus. Phosphorylation of Y8 disrupts inhibition, and there is evidence that in vivo glycolysis levels in erythrocytes are regulated in part by a phosphorylation/dephosphorylation signaling pathway. The structural basis for control by phosphorylation has been investigated by NMR studies on a complex between aldolase and a synthetic peptide corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this band 3 peptide (B3P) when it is bound to rabbit muscle aldolase was determined using the exchange-transferred nuclear Overhauser effect (ETNOE). Two hundred NMR structures for B3P were generated by simulated annealing molecular dynamics with NMR-derived distance restraints and excluding electrostatic terms. Twenty structures were further refined against a force field including full partial charges. The important conformational feature of B3P in the bound state is a folded loop structure involving residues 4-9 and M12 that surrounds Y8 and is stabilized by a hydrophobic cluster with the ring of Y8 sandwiched between the methyl groups of L4 and M12. Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, while terminal regions are structurally heterogeneous. To better understand B3P inhibition of aldolase and the mechanism of phosphorylation control, a complex was modeled by docking B3P into the active site of aldolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site residues. Binding is electrostatic in nature with numerous ionic and hydrogen-bonding interactions involving several conserved lysine and arginine residues of aldolase. How phosphorylation of band 3 could disrupt inhibition was considered by modeling a phosphoryl moiety onto Y8 of B3P. An energetic analysis with respect to rigid phosphate rotation suggests that aldolase inhibition is reversed primarily because of electrostatic repulsion between B3P residues that destabilizes the B3P loop formed in the complex. This proposed intramolecular mechanism for blocking protein--protein association by electrostatic repulsion with the phosphoryl group may be applicable to other protein--protein signaling complexes.
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PMID:Solution structure of a band 3 peptide inhibitor bound to aldolase: a proposed mechanism for regulating binding by tyrosine phosphorylation. 852 30

Glucose and phosphorus metabolism in mature (8-month-old) rat lenses were examined with NMR spectroscopy. Nondiabetic mature lenses contained sorbitol-3-phosphate (S3P) and fructose-3-phosphate (F3P) which were absent from young (1- to 2-month-old) normal rat lenses. The concentrations of these two phosphates can be changed through (1) diabetes induction with streptozotocin - this results in a dramatic increase in both compounds; and (2) oral dosing with a drug known to prevent sorbitol production - both metabolites disappeared. When normal mature lenses were incubated in 35.5 mM 13C1-glucose, both 13C1-lactate and 13C3-lactate were produced. Preservation of the 13C label at C1 is likely through the formation of 13C1-S3P and -F3P, which were then split through an aldolase-like mechanism into two 3-carbon compounds, one an unlabeled glycerol and the other 13C1-alpha-glycerophosphate (from S3P) and 13C1-dihydroxyacetone phosphate (from F3P). These reactions can contribute to the increase in alpha-glycerophosphate observed in both the streptozotocin-induced diabetic lenses and lenses incubated in high glucose.
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PMID:The further metabolism of sorbitol-3-phosphate and fructose-3-phosphate in the mature rat lens. 872 78

The generation of 13C-labeled D-glucose isotopomers by rat hepatocytes incubated for 30 or 120 min in the presence of 10 mM [3-(13)C]pyruvate was assessed by 13C NMR. The amount of C1-labeled D-glucose exceeded that of C2-labeled hexose, which was itself higher than that of C3-labeled D-glucose. A comparable hierarchy was observed in the C6-C5-C4 moiety of the hexose. The latter moiety of D-glucose was more efficiently labeled, however, than the C3-C2-C1 moiety. This finding is similar to that both previously reported and again observed in the present study when hepatocytes were exposed to [2(-13)C]pyruvate. These converging observations thus support the concept of enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phospho-fructoaldolase.
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PMID:Asymmetrical labeling of D-glucose generated from [3(-13)C]pyruvate in rat hepatocytes. 925 88

A protein-protein association regulated by phosphorylation of tyrosine is examined by NMR structural studies and biochemical studies. Binding of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and aldolase to the N-terminus of human erythrocyte anion transporter, band 3, inhibits enzyme activity. This inhibition is reversed upon phosphorylation of band 3 Y8, as shown by kinetic studies on purified components, as well as in vivo studies. Thus, tyrosine phosphorylation mediates against the intermolecular protein-protein association, in contrast to the positive control involving SH2 and PTB domains where phosphorylation is required for binding. To elucidate the basis of recognition and negative control by tyrosine phosphorylation, the structure of a synthetic peptide, B3P, corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2), bound to G3PDH has been determined using the exchange-transferred nuclear Overhauser effect. The G3PDH-bound B3P structure was found to be very similar to the structure recognized by aldolase. A hydrophobic triad forms from side chains within a loop structure of residues 4 through 9 in both bound species. Another structural feature stabilizing the loop, in the case of the B3P-G3PDH complex, is a hydrogen bond between the side chains of Y8 and D10 associated with a beta-turn of residues 8-11. Based on the structure of this phosphorylation sensitive interaction (PSI) loop, it is suggested that tyrosine phosphorylation disrupts protein-protein association, in part, by intramolecular electrostatic destabilization. The inhibition by B3P is competitive with respect to the coenzyme NAD+ and noncompetitive with the substrate analog arsenate. Specific binding of B3P to G3PDH is demonstrated by reversion of the NMR spectral properties of bound B3P to those of the free peptide upon addition of coenzyme and substrate analog. The stoichiometry of binding for the B3P-G3PDH complex was determined from Sephadex G-50 displacement experiments to be 4:1. Collectively, these results are consistent with B3P binding the active site of G3PDH.
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PMID:Insights into tyrosine phosphorylation control of protein-protein association from the NMR structure of a band 3 peptide inhibitor bound to glyceraldehyde-3-phosphate dehydrogenase. 945 76

Amide hydrogen exchange rates, determined by NMR spectroscopy, have become an important tool that is often used to investigate structure and dynamics of small proteins. Recent developments in mass spectrometry and sample handling methods make possible measurement of deuterium levels at peptide amide linkages in polypeptides. The ability to make these measurements has led to development of the protein fragmentation/mass spectrometry approach for determining amide hydrogen exchange rates in short segments of intact proteins following their incubation in D2O. Partially deuterated proteins are proteolytically fragmented into peptides whose molecular weights are determined by on-line liquid chromatography/mass spectrometry. Deuterium levels, which are determined from the molecular weights of the peptic fragments, can be used to determine amide hydrogen exchange rates. Details of the protein fragmentation/mass spectrometry approach, along with a brief review of the theory of amide hydrogen exchange, are described. The ability to detect and locate minor structural differences in proteins by the protein fragmentation/mass spectrometry approach is illustrated using oxidized and reduced cytochrome c. These results show that oxidation of iron has little effect on the N- and C-terminal regions, but significantly destabilizes the interior regions of cytochrome c. The ability to detect localized unfolding in large proteins is illustrated with aldolase that was equilibrated in acid. Despite the success achieved by NMR spectroscopy for determining amide hydrogen exchange rates, mass spectrometry is advantageous because it permits studies of large proteins, requires only picomoles of protein, and provides a direct measure of structural heterogeneity.
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PMID:Local structure and dynamics in proteins characterized by hydrogen exchange and mass spectrometry. 952 25

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.
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PMID:Metabolism of the dimethyl ester of [2,3-(13)C]succinic acid in rat hepatocytes. 987 64

A structural basis for activation and substrate specificity of src tyrosine kinases, and regulation of protein-protein association by tyrosine phosphorylation is described. Lyn, a src-family tyrosine kinase, recognizes and phosphorylates the immunoreceptor tyrosine-based activation motif, ITAM, a critical component in transmembrane signal transduction in hemopoietic cells. The structure of an ITAM peptide substrate bound to an active form of Lyn tyrosine kinase was determined by high-resolution NMR, and a model of the complex was generated using the crystallographic structure of Lck, a closely related Src-family kinase. The results provide a rationale for the conserved ITAM residues and specificity of Lyn, and suggest that substrate plays a role in stabilizing the kinase conformation optimal for catalysis. It is our hope that the Lck-ITAM peptide model complex will be useful in aiding structure-based drug design efforts that target substrate binding determinants in the design. Concerning the regulation of protein-protein association, we report on a complex between erythrocyte band 3 and two glycolytic enzymes, aldolase and glyceraldehyde-3-phosphate dehydrogenase. The formation of this complex is negatively regulated by tyrosine phosphorylation of band 3 by p72syk tyrosine kinase. In red blood cells, this association results in a decrease in glycolysis due to competitive inhibition of the glycolytic enzymes. The structure of band 3 recognized by the glycolytic enzymes was determined by solution NMR, and found to be a loop structure with tyrosine centrally positioned and excluded from intermolecular contact. This phosphorylation sensitive interaction, or PSI, loop may be the basis of a general mechanism for negative regulation through tyrosine phosphorylation.
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PMID:NMR structure of phospho-tyrosine signaling complexes. 1039 26

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.
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PMID:Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose. 1183 54

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.
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PMID:Biosynthesis of tetrahydrofolate. Stereochemistry of dihydroneopterin aldolase. 1203 64


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