Aldolase inactivation

To determine the kinds of residues modified, the aldolase inactivated with 1-[ C] bromopyruvate was first reduced, in the presence of a protein denaturant, with sodium borohydride to convert the ketone group of the incorporated pyruvyl moiety to a hydroxyl group. This was necessitated by the decarboxylation of the pyruvyl moiety (and therefore loss of the C label) during hydrolysis of the protein. ... 

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

These observations are explained by the mechanism shown in the figure. NaBH4 inactivates Class I aldolases by transfer of a hydride ion (H ) to the imine carbon atom of the enzyme-substrate adduct. The resulting secondary amine is stable to hydrolysis, and the active-site lysine is thus permanently modified and inactivated. NaBH4 inactivates Class I aldolases in the presence of either dihydroxyacetone-P or fructose-1,6-bisP, but inhibition doesn t occur in the presence of glyceraldehyde-3-P. 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

Hansen BA, Dekker EE. 1976. Inactivation of bovine liver 2-Keto-4-hydroxyglutarate aldolase by cyanide in the presence of aldehydes. Biochemistry 15 2912-2917. 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

Write a step-by-step sequence showing the chemical mechanisms involved in the action of a type I aldolase that catalyzes cleavage of fructose 1,6-bisphosphate. The enzyme is inactivated by sodium borohydride in the presence of the substrate. Explain this inactivation. 

We have developed new reaction systems based on colloidal dispersions [23, 24], namely highly concentrated water-in-oil (gel) emulsions, which could overcome most of the disadvantages of the aqueoussolvent mixtures such as inactivation of the aldolase and incomplete aldehyde solubilization in the medium. These emulsions are characterized by volume fractions of dispersed phase higher than 0.73 [25] therefore, the droplets are deformed and/or polydisperse, separated by a thin film of continuous phase. Water-in-oil gel emulsions of water/Ci4E4/oil 90/4/6 wt%, where C14E4 is a technical grade poly(oxyethylene) tetradecyl ether surfactant, with an average of four moles of ethylene oxide per surfactant molecule and oil can be octane, decane, dodecane, tetradecane, hexadecane, or squalane, were typically chosen as reaction media [23, 26]. 

As mentioned, the mildness of NaBHaCN (coupled with its effectiveness and stability in aqueous media) has attracted considerable interest for applications in biochemical areas. Examples include the trapping of suspected imine intermediates produced in enzyme (mitochondrial monoamine oxidase) inactivation by amines, the establishment by reduction of the positions of imine-forming amines in 2-keto-3-deoxy-6-phosphogluconate aldolase, and the transfer labeling of methionyl-tRNA synthetase and methionyl-tRNA transformalase by treatment with periodate-treated tRNA. In fact, most biochemical applications of NaBHaCN have utilized in situ imine formation-reduction (i.e. reductive amination) conditions and will be further discussed in Section 1.2.2.3.1. 

Evidence for the binding of protective molecules to important cellular macromolecules continues to appear. The protection of GED against inactivation of trypsin, lysozyme, and aldolase was considered not due to radical scavenging, but to mixed disulfide formation. Protection of DM by diamino disulfides was attributed to bound disulfide and protection of lactate dehydrogenase and catalase by serotonin was also attributed to complex formation, possibly with the metal ions in the enzymes. 

Aldolase is named for the mechanism of the forward reaction, which is an aldol cleavage, and the mechanism of the reverse reaction, which is an aldol condensation. The enzyme exists as tissue-specific isoenzymes, which all catalyze the cleavage of fructose 1,6-bisphosphate but differ in their specificities for fructose 1-P. The enzyme uses a lysine residue at the active site to form a covalent bond with the substrate during the course of the reaction. Inability to form this covalent linkage inactivates the enzyme. 

In our study on the substrate specificity of FDP aldolase, the compound 3-deoxy-3-fluorohydroxyacetone-l-phosphate was found not a substrate for the enzyme it was an inhibitor for the enzymatic cleavage of fructose-1, 6-diphosphate. The was determined to be 3 mM (Figure 4). When sodium borohydride was added to the mixture of aldolase and 3-deoxy-3-fluorohydroxyacetone-l-phosphate, no inactivation of aldolase was observed, indicating that a Schiff base is not formed. The Schiff base intermediate in the mixture of DHAP and aldolase has previously been trapped by reduction with sodium borohydride to inactivate the enzyme (8). The inhibitor may be useful for study of the active site of the enzyme. 

Some results indicate that different attempts of FucA immobilization by covalent attachment provoked severe enzyme inactivation (Fessner and Walter 1997). FucA and DERA from E. coli and SHMT from Streptococcus thermophilus have been immobilized by multipoint covalent attachment to glyoxyl-agarose. Although this immobilization method had been very successful with many different enzymes (Guisdn et al. 1993), results obtained with these aldolases were dissimilar. For FucA, in spite of an immobilization yield of 80-90%, enzyme inactivation occurred during immobilization process and only 10-20% of activity was retained (Suau et al. 2005). On the other hand, SHMT immobilization yield was 100%, but the immobilized activity was lost during the sodium borohydride reduction step, probably due to the reduction of the Schiff base established between the cofactor (pyridoxal phosphate) and the aldolase. Finally, 100% of immobilization yield and 65% of retained activity in the immobilized derivative was achieved with DERA. 

Aldolase then catalyses the reversible cleavage of the six-carbon molecule into two three-carbon molecules, triose phosphates. Yeast aldolase is inactivated by cysteine and may be reactivated by Zn +, Fe + or Co + ions. The triose phosphates are a mixture of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. Only the latter undergoes further change in the EMP pathway, but an equilibrium between the two is maintained by enzymic conversion of some of the dihydroxyacetone phosphate into glycer-aldehyde-3-phosphate, catalysed by the enzyme triose-phosphate isomerase. 

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