Carbonyl-lysine condensation

Aldolases such as fructose-1,6-bisphosphate aldolase (FBP-aldolase), a crucial enzyme in glycolysis, catalyze the formation of carbon-carbon bonds, a critical process for the synthesis of complex biological molecules. FBP-aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate (DHAP) and glyceralde-hyde-3-phosphate (G3P) to form fructose-1,6-bisphosphate. There are two classes of aldolases the first, such as the mammalian FBP-aldolase, uses an active-site lysine to form a Schiff base, whereas the second class features an active-site zinc ion to perform the same reaction. Acetoacetate decarboxylase, an example of the second class, catalyzes the decarboxylation of /3-keto acids. A lysine residue is required for good activity of the enzyme the -amine of lysine activates the substrate carbonyl group by forming a Schiff base. 

For the proline- and proline congener-catalyzed aldol reaction [23, 24], a mechanism based on enamine formation is proposed [25], Scheme 7. The catalytic process starts with condensation of the secondary amino group of proline with a carbonyl substrate leading to a nucleophilic enamine intermediate, which mimics the condensation of the active-site lysine residue with a carbonyl substrate in type I aldolases. The adjacent carboxylic acid group of the enamine intermediate... 

The free carbonyl residue can further condense with a histidyl group of another polypeptide chain to give a more complex cross-link. Schiff base formation, illustrated in Figure 8.4, involves an allysine residue at either the C- or N-terminal telopeptide and an uncharged lysine or hydroxylysine residue of another properly juxtaposed tropocollagen molecule. Such bonds are therefore intermolecular. Several other types of covalent cross-links are possible in collagens. They almost always involve lysine, allysine, hydroxylysine, hydroxyal-lysine, or histidine. 

Numerous undesirable reactions that result in organoleptic, nutritional and functional deterioration may occur in food proteins during processing and storage. These include the non-enzymatic or Maillard reactions, transamidation condensation reactions with dehydroalanine forming crosslinks, and carbonyl amine interactions, all of which may involve the free e-amino group of lysine (11,23). To minimize these reactions a significant volume of work has been done on the protective modification of the e-NH2 of lysine by formylation, acetylation, propionylation (26) or reductive dimethylation (10,11). 

FDP Aldolase. The most extensively utilized class of enzymes for monosaccharide synthesis are the aldolases (E.C. sub-class 4.1.2.). This ubiquitous group of enzymes catalyzes reversible aldol reactions in vivo. Two major groups of aldolases exist type I aldolases, found primarily in higher plants and animals, catalyze aldol condensations by means of a Schiff base formed between an enzyme lysine e-amino group and the nucleophilic carbonyl group type II aldolases, found primarily in microorganisms, utilize a divalent zinc to activate the nucleophilic component (79). Approximately 25 aldolases have been identified to date (18),... 

In the event, we observed that the resonance assigned to the methylene group a to the ketone carbonyl, C-1 appeared as a singlet of increased intensity in the NMR spectrum while the resonances for the carbonyl carbon atom C-2 and C-methyl carbon atom C-3 were spin-coupled. This pattern was indicative, at first sight, of condensation of two acetate units to acetoacetate and subsequent incorporation of the C4 compound (possibly as its CoA ester) via condensation of the carbon atom C-2 with the lysine derived imine (14) and subsequent or concomitant loss of C-1. The incorporation pattern into N-methylpelletierine (23) and its reduction product (24) appeared to be compatible with the classical Mannich scheme of Robinson, Path Cl in Scheme 8. 

Scheme 11.5. A cartoon representation of the catalyzed (fructose-bisphosphate aldolase, EC 4.1.2.13) aldol-type condensation between glyceraldehyde 3-phosphate and dihydroxyacetone monophosphate to produce the six-carbon ketosugar fructose-1,6-bisphosphate. An active site lysine Lys-NH2 [" H3NCH2CH2CH2CH2CH(NH3 )C02 ] apparently serves as the catalyst through addition at the carbonyl followed by proton tautomerization.

This is the mechanism of catalysis by aldolases which occur in plant and animal tissues (lysine aldolases or class I aldolases). A second group of these enzymes often produced by microorganisms contains a metal ion (metallo-aldolases). This group is involved in accelerating retroaldol condensations through electrophihc reactions with carbonyl groups ... 

After weighing the different posibilities to counter the lack of equal labeling in carbons 3 and 6 of lysine, Strassman and Weinhouse 180) propose as the more likely synthetic mechanism, a condensation of an acetyl methyl carbon with the carbonyl carbon of a-ketoglutarate, similar to the condensation of acetyl CoA and oxalacetate to yield citrate. This reaction would form homocitric acid. Upon oxidation and decarboxylation of the latter there would be obtained a-ketoadipic acid. Transamination of a-ketoadipic acid produces a-aminoadipic acid, which can be converted to lysine by reduction to the corresponding semialdehyde, followed by transamination. 

A primary amino group in the enzyme (supplied by the amino acid lysine. Section 26-1) condenses with the carbonyl group of the monophosphate este of 1,3-dihydroxy acetone to form an iminium ion. This species... 

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