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)

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.
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PMID:Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase. 0 53

Fructose 1,6-bisphosphate aldolase inactivation by L- and D-glyceraldehyde 3-phosphate (Ga 3-P) obeys pseudo first-order kinetics. L-Ga 3-P is much more effective than the D-isomer: the Ki values obtained are 0.032 mM and 0.54 mM respectively. Kinetic analysis suggests that one residue of the active center region is involved in the inactivation mechanism: specifically, a cysteine residue appears to be responsible for the initial inactivation by L-Ga 3-P. Lysine and arginine residues become involved at further steps of the inactivation mechanism. No correlation between loss of thiol groups and decay of catalytic activity was observed for the enzyme treated with D-Ga 3-P. The role of lysine and arginine residues in this reaction is discussed.
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PMID:Comparative aspects of the inactivation of fructose 1,6 bisphosphate aldolase by D- and l-glyceraldehyde 3-phosphate. 12 96

Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.
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PMID:Paracatalytic modification of aldolase: a side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues. 28 42

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
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PMID:Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver. 53 4

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.
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PMID:Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus. 56 49

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 x 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.
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PMID:Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase? 77 76

1. Treatment with methyl acetimidate was used to probe the topography of the tetrameric fructose 1,6-diphosphate aldolase from ox liver. A single treatment with imido ester in the presence or absence of 20mM-fructose 1,6-diphosphate caused the number of amino groups in the enzyme to fall to approx. 30% of the starting number (assumed to be 30 per subunit). The catalytic activity of the aldolase modified in the presence of fructose 1,6-diphosphate was unaffected, whereas that of the enzyme modified in the absence of substrate fell by about 20%. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that the amino group of lysine-27 (the numbering is that of the highly homologous rabbit muscle enzyme) is essentially unavailable for amidination in the native enzyme and is therefore predicted to be buried in a hydrophobic environment, probably in the form of an ion-pair with a negatively charged side-chain carboxyl group. All the other lysine residues that reacted poorly with methyl acetimidate in the native enzyme (a total of 7) were found to be within the primary structure bounded by lysine-107 and lysine-227. An important member of this group of lysine residues displaying aberrant reactivity is lysine-227, which is known to form an imine with the substrate as part of the catalytic mechanism of the enzyme. 3. The results of the amidination experiments can be correlated in an interesting way with previous studies of thiol-group modification in the aldolases. Taken together, and arguing in part by analogy with the results of identical experiments with glyceraldehyde 3-phosphate dehydrogenases where the three-dimensional structure is known [Lambert & Perham (1977) Biochem. 4. 161. 49-62], they suggest that the region of primary structure from residues 107-227 may form the whole or part of a three-dimensional structural feature, perhaps a folding domain. A three-dimensional structure deduced from X-ray-crystallographic analysis will be needed to interpret these findings more closely. 4. The amino groups of lysine residues are commonly thought to reside at the 'surface' of protein structures. The patterns of specific lysine residues in glyceraldehyde 3-phosphate dehydrogenases and in aldolases that have been found to react poorly with methyl acetimidate in the native enzymes can be attributed to intramolecular ionic interactions deep in hydrophobic pockets and at the protein 'surface'. Such ionic interactions may contribute significantly to the stability of a given protein.
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PMID:Intramolecular ionic interactions of lysine residues and a possible folding domain in fructose diphosphate aldolase. 85 25

Pyridoxal 5'-phosphate (PLP) in aqueous solutions can form a Schiff base complex with 14 and 16 lysine residues of rabbit and sturgeon muscle aldolases (EC 4.1.2.13), respectively. Although the mechanism of their interaction with PLP should be the same, these residues can be differentiated into three families on the basis of their inhibition constant Ki and rate constant k. The lysine residues of one of these families do not react with PLP in the presence of the substrates. Therefore, they are assumed to be part of the active center. In the sturgeon muscle aldolase, 3.7 substrate protected lysine residues are present. Rabbit aldolase, although tetrameric, contains only 2.8 substrate protected lysine residues. This suggests that one active center of this enzyme may be 'buried'. Structural studies showed the following sequence around the substrate protected lysine residues, in the rabbit aldolase: Gly-(Gly2, Val3)-Pyridoxyl Lys-Ile-Asp-Lys.
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PMID:Differential effects of pyridoxal 5'-phosphate on lysine residues in rabbit and sturgeon muscle aldolases. 95 52

Lysinuric protein intolerance (LPI), an autosomal recessive defect of diamino acid transport, is characterized chemically by renal hyperdiaminoaciduria, especially lysinuria, and by impaired formation of urea with hyperammonemia after protein ingestion. Our 20 patients thrived during breast-feeding, but ingestion of cow's milk caused diarrhea and vomiting. When able to select their diet, they rejected all protein-rich foods. They were short staturated and had weak atrophic muscles, osteoporosis, hepatomegaly and often splenomegaly. Four patients were mentally retarded. Fifteen patients had leukocyte counts below 4,000/mm3, and 17 patients had platelet counts below 150,000/mm3. Serum lactate dehydrogenase activity was constantly increased, and transaminase and aldolase activities were often increased. In the infants' livers, changes were only revealed by electron microscopy: increased and vesicular smooth endoplasmic reticulum, and abundance of glycogen particles in the hepatocytes. In the older patients, light microscopy demonstrated clearly limited areas where hepatocytes had large pale cytoplasm and small pyknotic nuclei. The diamino acids lysine, arginine and ornithine had plasma concentrations only one-third to one-half the normal mean; the renal clearances were clearly increased. Oral diamino acid loading tests suggested impaired intestinal absorption. Urea is built in the liver through transformation of ornithine to arginine, and cleavage of arginine to ornithine and urea. The addition of ornithine to an intravenous I-alanine loading prevented the hyperammonemia and normalized the urea production. Therefore, the diet has been supplemented with arginine, and more protein has been added. This therapy has lead to a remarkable catch-up growth in some patients. The pathophysiology of LPI is explained. Because of defective intestinal absorption and incrased renal loss, the diamino acids have a low plasma concentration. Their transport from plasma to hepatocytes is also impaired, and the liver becomes deficient in ornithine. This retards the urea cycle, and leads to postprandial hyperammonemia and protein aversion. The presence of the transport defect in the hepatocytes distinguishes LPI from other hyperdibasicaminoacidurias.
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PMID:Lysinuric protein intolerance. 115 80

We investigated three aldolase isozymes (aldolase A, B, and C) in human lung cancer by using an indirect peroxidase labeled antibody method. We used 27 tissue samples obtained at surgical operations which were fixed in periodate-lysine-4% paraformaldehyde (PLP) solution, and embedded in optimum cutting temperature (OCT) compound. They were 11 adenocarcinomas, 9 squamous cell carcinomas, 3 large cell carcinomas, 3 small cell carcinomas, and 1 adenosquamous carcinoma. Aldolase A and C expressed intensely positive stainings in the cytoplasm of cancer cells compared with normal lung tissues, and its positivities were 81% respectively. However, Aldolase B showed almost negative staining, and its positivities were only 41%. These rates had no relation to the histological types or pathological stages of lung cancers, and suggested that human lung cancer contained increased levels of aldolase A, and C.
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PMID:[An immunohistochemical study on three aldolase isozymes in human lung cancer]. 131 19


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