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The mechanism of action of aldolase and the asymmetric labeling of hexose
... 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 ...
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.
This chapter discusses the several aspects of the formation of shikimate, and with its conversion to aromatic amino acids. Although the role of D-glucose is stressed in the title, it should be pointed out that pyruvate and acetate, the starting materials for the other known pathways, may also be derived ultimately from D-glucose. A bacterial culture in a minimal medium (salts plus D-glucose) was irradiated with ultraviolet light, and the resultant mixture of wild-type and mutant strains was grown on a medium supplemented with yeast extract and casein hydrolyzate. Mutant strains were obtained which required for growth phenylalanine, tyrosine, tryptophan, and p-aminobenzoic acid. For certain of these strains, it was found that, of more than 50 compounds tested, only shikimic acid was able to replace the required aromatic supplement; other strains accumulated shikimic acid in the culture medium. It was therefore concluded that the former can convert shikimic acid to the aromatic metabolites, the genetic block in these organisms being somewhere before shikimic acid; the latter clearly were blocked after shikimic acid. Certain multiple aromatic auxotrophs grew slowly on the quadruple aromatic supplement, but were stimulated by the further addition of a trace of shikimic acid or of wild-type filtrate. The compound responsible for this effect was shown to be p-hydroxybenzoic acid, a bacterial vitamin subsequently found to be involved in the biosynthesis of methionine and lysine.
: The synthetic polymeric catalysts containing pendent imidazole groups have been studied. Poly-4(5)-vinylimidazole and poly-5(6)-benzimidazole exhibited multifunctional catalyses, similar in mechanism to those of an enzyme in the solvolyses of positively or negatively charged esters or a neutral ester. The cooperative interactions between the imidazole moiety and other functional groups such as carboxyl, hydroxyl and phenol have been also observed in the solvolyses of esters. The nature of these interactions is discussed. More detailed studies are in progress. An attempt was made to ascertain the stereochemical selectivity of the polymeric catalyst. An optically active alternating copolymer of 4(5)-vinylimidazole and an optically active vinylketone did not exhibit selectivity. Another type of an optically active polymeric imidazole will be investigated. Steric requirements seem to be an important factor in the polymeric imidazole catalysts. Poly-2-vinylimidazole had a very low catalytic activity when compared with poly-4(5)-vinylimidazole. The synthesis of a new polymeric catalyst poly-3-vinyl-1,2,4-triazole is in progress. (Author)
Many proteins have been crystallized and enzymatically identified from the aqueous extract of the rabbit muscle than from any other material. The methodology of enzyme preparation has been improved. The investigation at the molecular level of enzyme mechanisms has also been greatly stimulated. This stimulus is now extended with the aim of a better understanding of the cellular physiology of these well-defined muscle constituents. Myogen, a mixture of proteins, is the major component of juice, which can be mechanically pressed from skeletal muscle. It is extracted at higher yields, with somewhat altered proportions of the constituents by water or by salt solutions, with an ionic strength not exceeding the ionic strength of muscle juice. High-salt concentrations extract myogen together with the proteins of the myosin and actin group; the latter two being resistant to low ionic strength extractions because of poor solubility or structural inaccessibility.
Octanoate and N6,O2'-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) cause a marked inhibition of net glucose utilization and lactate and pyruvate accumulation by hepatocytes isolated from meal-fed rats. Acetate is much less effective as an inhibitor of glycolysis. Fatty acid synthesis, as measured by tritiated water incorporation, is inhibited by dibutyryl cyclic AMP, whereas it is stimulated by 10 mM acetate and 1 mM octanoate. Stimulation of fatty acid synthesis by 1 mM octanoate, however, is lost paradoxically at higher concentrations of octanoate. Rates of fatty acid synthesis estimated by [1-14C]octanoate incorporation were consistently higher than rates calculated on the basis of tritiated water incorporation, raising the question as to which is the better index of the rate of de novo fatty acid synthesis. The effects of octanoate were studied because it was reasoned that this fatty acid should not inhibit acetyl-CoA carboxylase but should inhibit glycolysis and supply acetyl-CoA for lipogenesis. This was found to be the case, proving that glycolytic activity is not necessary for rapid rates of de novo fatty acid synthesis by liver.
A tryptic peptide has been isolated from the active site of fructose 1,6-diphosphate aldolase and its amino acid sequence has been determined. Crystalline enzyme preparations were labeled by reduction of the Schiff base intermediate with dihydroxyacetone phosphate-14C, resulting in the formation of approximately three equivalents of N6-β-glyceryllysine per mole of enzyme. The protein was S-carboxymethylated and digested with trypsin, and the 14C-labeled peptides were isolated by ion-exchange chromatography and electrophoresis. The sequence of the major peptide was: Ala-Leu-AsN-AsN-His-His-Val-Tyr-Leu-Ser-GlN-Gly-Thr-Leu-Leu-βGLys-AsN-Pro-Met-Val-Thr-Ala-Gly-His-Ala-CMCys-Thr-Lys. This peptide is very similar in structure to the corresponding peptide from rabbit muscle aldolase. There is a striking conservation of amino acids adjacent to the reactive lysine residue, which may indicate a structure necessary for the specific function of the enzyme.
IntroductionMetalloaldolases (Class II)Schiff Base-Forming Aldolases (Class I)Mammalian Aldolases
Several ligands, including fructose 1,6-diphosphate, and substrate analogues, such as hexitol diphosphate, are effective in protecting muscle aldolase against digestion by carboxypeptidase. Several lines of evidence suggest that the conformational change affecting the carboxyl-terminal tyrosine residues is mediated by interaction of the ligands with the 6-phosphate binding site of the enzyme. (1) The dissociation constants for phosphate esters, based on protection against the action of carboxypeptidase, were found to be comparable to the values in the literature for the dissociation constant for phosphate at the 6-phosphate binding site of the enzyme. (2) β-Glycerophosphate aldolase is as sensitive to carboxypeptidase as is the native enzyme, and both are protected to the same extent by hexitol diphosphate or inorganic phosphate against digestion by carboxypeptidase. (3) The modified enzyme formed when aldolase is allowed to react with glyceraldehyde 3-phosphate in the absence of dihydroxyacetone phosphate is markedly resistant to digestion by carboxypeptidase.Support is provided for a reaction mechanism in which the 3-phosphate group of glyceraldehyde 3-phosphate (or 6-phosphate of fructose 1,6-diphosphate) enhances the nucleophilic character of a group at the active center. The presence of the carboxyl-terminal tyrosine residue is essential for the integrity of this structure.The present results may serve to explain the anomaly which has existed between the number of subunits, the number of active sites, and carboxyl-terminal tyrosine residues in rabbit muscle aldolase.
Hepatic fructose-6-phosphate (fructose-6-P) cycling and pentose cycle activity were quantified in hyperthyroid patients. A measure of the fructose-6-P cycle was the incorporation of 14C, on administering [3-3H,6-14C]galactose, into carbon 1 of blood glucose and the 3H/14C ratio in blood glucose. The measure of the pentose cycle was the randomization of 14C to carbon 1 of blood glucose on administering [2-14C]galactose. [2-3H]Galactose was also administered, so the 3H/14C ratio in blood glucose measured the extent of equilibration of glucose-6-P with fructose-6-P. Patients given [3-3H,6-14C]galactose were restudied when euthyroid. Of the 14C from [3-3H,6-14C]galactose, 7.7-9.5% was in carbon 1 of glucose in both states. 3H/14C ratios were also the same in both states. Fructose-6-P cycling was estimated to be 13 +/- 1% the rate of glucose turnover in the euthyroid and 15 +/- 1% that in the hyperthyroid state. The pentose cycle contributed about 2% to glucose utilization, similar to previous estimates in healthy humans. As in healthy individuals, about 25% of 3H was retained in the conversion of [2-3H]glucose-6-P to glucose. Thus, the fractions of glucose turnover participating in hepatic fructose-6-P and pentose cycling are similar in hyperthyroid and healthy subjects. As a result, augmented fructose-6-P cycling does not substantially contribute to increased hepatic oxygen consumption in hyperthyroidism.
Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90.5 MHz. [1-13c]Glucose and [6-13C]glucose were fed to suspensions of yeast cells. Time courses for concentration changes of the starting material, of courses for concentration changes of the starting material, of the intermediate fructose 1,6-bisphosphate (Fru-P2), and of the end products, ethanol and glycerol, have been followed with 1-min time resolution. The glucose uptake was well fitted by a Michaelis-Menten model, assuming competition of alpha- and beta-glucose for the same site. The Km for the uptake was found to be 10 mM for beta-glucose and 5 mM for alpha-glucose. The concentration of Fru-P2 showed an initial oscillation before it reached a co,stant level. The 13C label, introduced only as [-13C]- or [6-13C]glucose, was observed in Fru-P2 in both the C1 and C6 positions, simultaneously. From the relative intensities of the C1 Fru-P2 and C6 Fru-P2 peaks in the presence of [1-13C]- and [6-13C]glucose, in vivo kinetic information was obtained about the aldolase-triosephosphate isomerase triangle. We found that under the conditions of these experiments the ratios of backward to forward velocities through aldolase and triosephosphate isomerase were 0.9 +/- 0.1 and 0.8 +/- 1, respectively, indicating they were close to equilibrium.
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
A convenient method has been developed for determination of the label distribution within the anhydro-D-glucose units of C14-labeled cellulose. The method is based on the periodate oxidation of cellulose followed by further oxidation of the resulting aldehyde functions with chlorous acid and hydrolysis of the product to glyoxylic and D-erythronic acids. The former compound was isolated as the 2,4-dinitrophenylhydrazone, which on decarboxylation provided the label distribution at C1 and C2. Complete and partial periodate oxidation of D-erythronic acid, isolated as the lactone, gave the label at C3, C4, C5 and C6. Application of this method to a sample of cotton cellulose-C14, prepared from D-glucose-2-C14, indicated that 58% of the label had remained at C2 and 25% was transferred to C5. These data and the distribution of label in the cotton seed oil indicate that such breakdown and resynthesis of D-glucose as occurs, takes place through the Embden-Meyerhof pathway.
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-C¹ⴠ; were administered orally to rats and the distribution of C¹ⴠin 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 C¹ⴠin ; 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 glycerolC¹â´. 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);
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.
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.
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).
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.
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.