Aldolase enzyme-substrate complex

Treatment with sodium borohydride of the enzyme-substrate complex of aldolase A and dihydroxyacetone phosphate leads to formation of a covalent linkage between the protein and substrate. This and other evidence suggested a Schiff base intermediate (Eq. 13-36). When 14C-containing substrate was used, the borohydride reduction (Eq. 3-34) labeled a lysine side chain in the active site. The radioactive label was followed through the sequence determination and was found on Lys 229 in the chain of 363 amino acids.186/188 188b Tire enzyme is another (a / P)8-barrel protein and the side chain of Lys 229 projects into the interior of the barrel which opens at the C-terminal ends of the strands. The conjugate base form of another lysine,... 

There are two distinct classes of the enzymes known as FDP (fructose 1,6-diphosphate) aldolases, which carry out an important and early stage in glycolysis (a), the varieties found in animals and higher plants cleave FDP byway of a Schiff base, whereas (b), those which occur in bacteria and fungi require a metal (usually Zn " ") bound to the carbonyl group in the enzyme-substrate complex special inhibitors exist for the second variety (Lewis and Lowe, 1973). More examples of differing metal requirements will be found in Section 11.1 see under Helminths in Section 4.4 for some other differences in analogous enzymes. 

Aldolase antibodies obtained by reactive immunization are notable for high activity, broad substrate specificity, and high selectivities [53]. Rate accelerations are typically in the range 105 to 107-fold over background. Although the k /K values are 102 to 104 lower than those of aldolase enzymes, these are among the most efficient antibody catalysts described to date. Their efficacy is all the more notable in light of the inherently complex, multistep process they catalyze. 

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. 

As well as complexing the substrate to the active site, many enzymes link covalently with the substrate, or a portion of it, to form an additional intermediate. Such intermediates occur in the action of enzymes as diverse as alkaline phosphatase (phosphoryl enzyme), serine and cysteine proteases (acyl enzymes), glycosidases (acylal enzymes) and aldolases. 

K ion is a cofactor for the aldolase reaction. Pyruvic kinase from all known sources also requires univalent cations, in addition to a divalent cation. This enzyme needs K, ammonium, or Rb ions as a cofactor. Mg ions serve an important function in photosynthetic processes in tobacco as it is an essential constituent of chlorophyll a and chlorophyll b. Some heavy metal and nonmetal ions are toxic to tobacco and can serve as metabolic inhibitors. The toxicity of fluoride, for example, is explained in part on the basis of the formation of a magnesium-fluorphosphate complex that inhibits the eno-lase reaction in glycolysis. Other enzymes are inhibited by substrate analogs, sulfhydryl complexing agents, and metal chelating agents. 

Nuclear relaxation studies of substrates and inhibitors have resulted in the detection of 10 enzyme-Mur-substrate and 4 enzyme-Mn-inhibitor bridge complexes possessing kinetic and thermodynamic properties consistent with their participation in enzyme catalysis. Three cases of a activation, by divalent cations, of enzyme-catalyzed enolization reactions (pyruvate carboxylase, yeast aldolase, v-xylose isomerase), and one case of 8 activation of an enzyme-catalyzed elimination reaction (histidine deaminase) have thereby been established, Thus, in each proven case, the enzyme-bound Mn coordinates an electronegative atom (Z) of the substrate, which is attached to a carbon atom one or two bonds away from the carbon atom which is to be deprotonated ... 

Following the study by Westheimer and Cohen [19] of the primary and secondary amine catalyzed dealdolization of diacetone alcohol which was proposed to occur through an essential imine intermediate. Speck and Forist [28] suggested that the S2une class of reaction may occur in aldolase. The first characterization of a stable covalent entity formed between substrate and enzyme was done by Horecker et al. [29] on transaldolase. This enzyme, which forms a stable complex with dihydroxy-acetone formed from the cleavage of fructose-6-P, was found to be susceptible to reduction by sodium borohydride. It was demonstrated that this adduct was formed... 

From a synthetic point of view, aldolases offer a number of advantages as catalysts for C—C bond formation. For example, they operate best on unprotected substrates, thus avoiding the problem of complex protection/ deprotection schemes for polyfunctional molecules (e.g. carbohydrates). They also catalyse C—C bond synthesis with high diastereoselectivity and enantioselectivity. Such simultaneous control is often difficult to achieve using non-enzymic aldol reactions. 

For class I type enzymes, the (/ia)8-barrel structure of the class I fructose 1,6-bisphosphate aldolase (FruA, vide infra) from rabbit muscle was the first to be uncovered by X-ray crystal-structure analysis [33] this was followed by those from several other species [34-37]. A complex of the aldolase with non-covalently bound substrate DHAP (dihydroxyacetone phosphate) in the active site indicates a trajectory for the substrate traveling towards the nucleophilic Lys229 N [38, 39]. There, the proximity of side-chains Lysl46 and Glul87 is consistent with their participation as proton donors and acceptors in Schiff base formation (A, B) this was further supported by site-directed mutagenesis studies [40]. 

Structural details are also available for the class II (/ia)8-barrel enzyme FruA from E. coli at excellent resolution (1.6A) [51, 52]. The homodimeric protein requires movement of the divalent zinc cofactor from a buried position to the catalytically effective surface position. Recent attempts to explore the origin of substrate discrimination of the structurally related E. coli aldolase vith specificity for tagatose 1,6-bisphosphate by site-directed mutagenesis and structure determinations highlight the complexity of enzyme catalysis in this class of enzymes and the subtleties in substrate control [53-55]. 

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