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The mechanism of action of aldolase and the asymmetric labeling of hexose

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... dihydroxyacetone-P carbanion intermediate we showed that glyceraldehyde-3-Pi exchanged with FDP much faster than did DHAP at equilibrium. This appears to be the first time that relative rates of exchange at equilibrium were used to infer a mechanism (Rose, 1958). Paul ...
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A procedure for purifying N-acetylneuraminic acid aldolase of Clostridium perfringens is described. The purified enzyme has a molecular weight of 92,000 and consists of two protein components each of which has enzymatic activity. The purified aldolase has a pH optimum of 7.2 and a Km of 1.75 mm; heavy metal ions are potent inhibitors. No metal ion requirement could be demonstrated for the enzyme and it is completely inhibited by sodium borohydride reduction in the presence of N-acetylneuraminic acid. This indicates that the enzyme is a Class I aldolase and forms a Schiff base-enzyme complex. There is no requirement for a substrate carboxyl group for binding to occur; substitution of a polar or bulky group at the deoxy position causes decreased binding. Substrate analog binding did not result in effective cleavage of the appropriate carbon-carbon bond. Kinetic studies with the pyruvate analog bromopyruvate are consistent with the existence of nucleophilic residues within the active site.
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IntroductionMetalloaldolases (Class II)Schiff Base-Forming Aldolases (Class I)Mammalian Aldolases
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Chapter
IntroductionNomenclature of the Active SiteCovalent Labeling of the Active SiteIdentification of Amino Acids at the Active Site without Covalent LabelingAmino Acid Composition of the Active SiteThree-Dimensional Structure of the Active SiteRole of the Active SiteConclusion
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h deuterium in the stereospecific position which is removed by ; triosephosphate isomerase during conversion of dihydroxyacetone phosphate to ; glyceraldehyde 3-phosphate. Both deuterated and nondeuterated glycerol-C¹⁴ ; were administered orally to rats and the distribution of C¹⁴ in the hexose ; units of liver glycogen was investigated. It was established that the presence ; of deuterium in the glycerol causes an increased asymmetry of the C¹⁴ in ; the glucose unit. It is known that purified liver triosephosphate isomerase uses ; dihydroxyacetone phosphate less rapidly when it is deuterated at the position ; cleaved than tt does the nondeuterated dihydroxyacetone phosphate when the two ; species are mixed. Theoretical considerations indicate that discrimination ; against the deuterated species of dihydroxyacetone phosphate would be reflected ; in the labeling of the glyceraldehyde 3-phosphate and dihydroxyacetone phosphate ; and that the extent of the difference in labeling is dependent on the rate of the ; reversible interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone ; phos phate. It was shown that the labeling of the glucose is consistent with ; predictions for such discrimination. The results provide strong evidence for the ; noneqnilibration of the triosephosphate pools. It is concluded that failure to ; achieve isotopic equilibration at the triosephosphate stage is the explanation ; for the unequal labeling of the glucose units of liver glycogen derived from ; administered glycerolC¹⁴. The present experimental approach demonstrates ; the use of deuterium to alter rates of reactions to evaluate the extent of ; equilibration of metabolic intermediates under normal physiological conditions in ; vivo. (auth);
Chapter
Aldolase has been shown to be highly ubiquitous, occurring in all plant and animal tissues examined and in most microorganisms. It is absent in some heterofermentative bacteria such as Leuconostoc mesenteroides, and in some aerobic organisms such as Acetobacter suboxydans. On the basis of the difference in metal requirement, and other properties, aldolases have been classified into two categories—Class I and Class II. Class I aldolases, present in animals and higher plants, do not appear to require a metal ion cofactor and form a Schiff base intermediate with their substrate. These are inactivated by reduction with NaBH4 in the presence of the substrate. The Class II aldolases, found in bacteria and molds, are not inactivated by reduction with NaBH4 in the presence or absence of substrate. Certain blue-green algae appear to contain both types of aldolase. The molecular weight of Class II aldolases, where determined, is in the neighborhood of 70,000, approximately half that of mammalian aldolases, and they are composed of two, rather than four, subunits. The activity toward fructose-1, 6-bisphosphate (FDP) is not significantly affected by treatment with carboxypeptidase. Multiple forms of Class II aldolase have also been found in yeast and bacteria. Crystalline yeast aldolase can be resolved to yield three active components by isoelectric fractionation.
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In mammals and most other organisms, the first step in the metabolism of glucose is its phosphorylation to glucose-6-phosphate. The metabolic pathways of glucose-6-phosphate utilization can be conveniently divided into two major groups: a) pathways in which glucose is broken down to triose phosphate and will be designated as triose pathways ; and b) pathways that do not yield triose-phosphate and which will be designated here as non-triose pathways. Until fairly recently, it was generally held that glucose is catabolized to triose phosphate solely by the reactions of the classical Embden-Meyerhof (E. M.3) pathway. An alternate pathway of glucose phosphate catabolism in mammalian tissues has been known since about 1930 from Warburg’s discovery of the direct oxidation of glucose-6-phosphate. By 1936, it was known that the aldehyde carbon of glucose-6-phos-phate is decarboxylated to CO2 and a pentose phosphate is formed; and Dickens and Lipmann proposed schemes for glucose oxidation based on these observations. However, little attention was paid to these schemes and the mechanism of this pathway remained obscure until about 1950. Since that time, the detailed operation of this scheme has been elucidated by the work of several laboratories, especially those of Horecker and Racker. The pentose pathway yields CO2 and 1 mole of triose phosphate per mole of glucose, whereas the E. M. pathway yields two moles of triose phosphate, and the subsequent metabolism of the triose phosphate is common to both pathways.
Chapter
Lyases are enzymes that cleave C—C, C—O, C—N, and other bonds in such manner that the cleavage event does not involve hydrolysis or oxidation. In the cleavage direction the reaction is often upon one substrate only. During cleavage a molecule is eliminated from the substrate, leaving an unsaturated residue. The eliminated fragment is usually linked covalently to the (holo)enzyme during the cleavage process, and is ultimately transferred to an acceptor, often a proton. The decarboxylation of acetoacetate to acetone and carbon dioxide is a familiar example of a lyase reaction in which a C—C bond is cleaved. The present chapter gives a sampling of lyase reactions, showing the diverse chemical mechanisms by which they are (thought to be) catalyzed. Table 5.1 at the end of the chapter lists 56 lyases for which there is evidence of a covalent enzyme-substrate intermediate in their catalytic cycles. They comprise 23% of all the lyases recognized officially by the Enzyme Commission; and, on grounds of chemical analogy, they exemplify, in aggregate, over 90% of the EC lyases (cf. Chapter 8).
Chapter
Die Methode der Markierung von Verbindungen, welche bei der Aufklärung von Stoffwechselwegen im komplexen System lebender Zellen so große Fortschritte gebracht hat, kann auch sinngemäß auf einzelne Enzymreaktionen angewendet werden, indem man das Substrat an bestimmten Atomen radioaktiv markiert und im Reaktionsprodukt Umfang und Ort der Markierung bestimmt. In vielen Fällen stellt diese Methode nur eine Variation einer Bestimmungsmethode dar, die vor einer anderen quantitativen Bestimmung bei Vorhandensein geeigneter Apparate Vorteile hinsichtlich der Schnelligkeit, Spezifität und Empfindlichkeit aufweisen kann. Eine Markierung des Phosphors in einem organischen Phosphorsäureester kann z. B. benutzt werden, um eine Spaltung durch Phosphatase auch in Gegenwart größerer Mengen von Fremdphosphor verschiedener Bindungsweise quantitativ zu untersuchen. Auch die vielen Nachweise und Bestimmungsmethoden bei der Überführung von Phosphor in eine andere Bindungsweise sind hier zu nennen. Man bedient sich hierbei vorzugsweise der Papierchromatographie, Papierelektrophorese oder Ionenaustauschchromatographie zur Trennung der vorliegenden Gemische. Die phosphorhaltigen Reaktionsprodukte lassen sich dann durch Radioautographie leicht sichtbar machen.
Article
The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 A resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P1-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P1-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P1-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.