Aldolase, active lysine

Antibody Catalysis. Recent advances in biocatalysis have led to the generation of catalytic antibodies exhibiting aldolase activity by Lemer and Barbas. The antibody-catalyzed aldol addition reactions display remarkable enantioselectivity and substrate scope [18]. The requisite antibodies were produced through the process of reactive immunization wherein antibodies were raised against a [Tdiketone hapten. During the selection process, the presence of a suitably oriented lysine leads to the condensation of the -amine with the hapten. The formation of enaminone at the active site results in a molecular imprint that leads to the production of antibodies that function as aldol catalysts via a lysine-dependent class I aldolase mechanism (Eq. 8B2.12). 

Aldol condensation reactions are catalyzed by amines and the active sites of many aldolases contain an essential lysine residue. Using a strategy of reactive immunization with a 1,3-diketone (18 in Fig. 5.8), Wagner et al. were able to generate antibodies with aldolase activity. These were shown to possess a highly reactive lysine residue in... 

Similar peptide dendrons from the 2.2.2.2 series prepared in a library format were screened, while still on the beads, for potential aldolase activity.Probes forming colored enaminones or releasing fluorescent fragments via enolization or retro-aldol reactions were used for the screening. Lysine- and proline-rich sequences were identified with the enaminone-forming and enolization-sensitive probes. 

Two classes of aldolase enzymes are found in nature. Animal tissues produce a Class I aldolase, characterized by the formation of a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate. Class I aldolases do not require a divalent metal ion (and thus are not inhibited by EDTA) but are inhibited by sodium borohydride, NaBH4, in the presence of substrate (see A Deeper Look, page 622). Class II aldolases are produced mainly in bacteria and fungi and are not inhibited by borohydride, but do contain an active-site metal (normally zinc, Zn ) and are inhibited by EDTA. Cyanobacteria and some other simple organisms possess both classes of aldolase. 

FIGURE 19.13 (a) A mechanism for the fructose-l,6-bisphosphate aldolase reaction. The Schlff base formed between the substrate carbonyl and an active-site lysine acts as an electron sink, Increasing the acidity of the /3-hydroxyl group and facilitating cleavage as shown. (B) In class II aldolases, an active-site Zn stabilizes the enolate Intermediate, leading to polarization of the substrate carbonyl group. 

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. 

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedohep-tulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine hydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate. 

The active site of the aldolase enzyme is believed to be as shown (Figure 13.7). Although several amino acid residues are involved with bonding the substrates at the active site, the critical amino acid residues are a lysine and an aspartic acid residue. The lysine forms a substrate-enzyme bond via an imine linkage, and the aspartic acid residue functions as a general acid-base. 

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

The structure of dihydroneopterin aldolase has been analyzed further with respect to the functional roles of conserved active site glutamate and lysine residues <2006B15232>. NMR studies have also suggested that the isomerization of dihydroneopterin to dihydromonapterin catalyzed by the same enzyme involves the action of the same functional groups <2007MI2240>. 

Aldolases have been classified into mechanistically distinct classes according to their mode of donor activation. Class 1 aldolases achieve stereospecific deprotonation via covalent imine/enamine formation at an active-site lysine residue, while Class II aldolases utilize a divalent transition metal cation for substrate coordination as an essential Lewis acid cofactor (usually Zn ) to facilitate deprotonation... 

The first step in this sequence is the binding of a molecule of acetaldehyde ( donor ) to the aldolase to form a Schiff base with the active site lysine followed by addition to CIAA, which acts as the acceptor aldehyde. This reaction delivers the mono-addition product, which then acts as an acceptor again to react with a second molecule of AA, yielding the double addition product which cyclizes spontaneously to the stable lactol 1 (Scheme 6.4). 

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,... 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysine s-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily in microorganisms and utilize a divalent zinc to activate the electrophiUc component of the reaction. The most studied aldolases are fructose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn2+-containing aldolase from E. coll In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetonephosphate [57-04-5] (DHAP). 

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. 

The new aldolase differs from all other existing ones with respect to the location of its active site in relation to its secondary structure and still displays enantiofacial discrimination during aldol addition. Modification of substrate specificity is achieved by altering the position of the active site lysine from one /3-strand to a neighboring strand rather than by modification of the substrate recognition site. Determination of the 3D crystal structure of the wild type and the double mutant demonstrated how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate. It is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. 

In proteins with a symmetric structure, circular permutation can account for the shift of active-site residues over the course of evolution. A very good model of symmetric proteins are the (/Ja)8-barrel enzymes with their typical eightfold symmetry. Circular permutation is characterized by fusion of the N and C termini in a protein ancestor followed by cleavage of the backbone at an equivalent locus around the circular structure. Both fructose-bisphosphate aldolase class I and transaldolase belong to the aldolase superfamily of (a/J)8-symmetric barrel proteins both feature a catalytic lysine residue required to form the Schiff base intermediate with the substrate in the first step of the reaction (Chapter 9, Section 9.6.2). In most family members, the catalytic lysine residue is located on strand 6 of the barrel, but in transaldolase it is not only located on strand 4 but optimal sequence and structure alignment with aldolase class I necessitates rotation of the structure and thus circular permutation of the jS-barrel strands (Jia, 1996). 

The formation of covalent substrate-catalyst adducts might occur, e.g., by single-step Lewis-acid-Lewis-base interaction or by multi-step reactions such as the formation of enamines from aldehydes and secondary amines. The catalysis of aldol reactions by formation of the donor enamine is a striking example of common mechanisms in enzymatic catalysis and organocatalysis - in class-I aldolases lysine provides the catalytically active amine group whereas typical organocatalysts for this purpose are secondary amines, the most simple being proline (Scheme 2.2). 

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... 

In our original hapten design for aldolase antibodies, the /3-diketone functionality of hapten 4 was used as a reactive immunogen to trap a chemically reactive lysine residue in the active site of an antibody as a stable enaminone. The chemical mechanism leading up to the stabilized enaminone should match that of Class I aldolases over this portion of the reaction coordinate. 

Mechanistically, the antibody aldolases resemble natural class I aldolase enzymes (Scheme 4.7) [52]. In the first step of a condensation reaction, the s-amino group of the catalytic lysine reacts with a ketone to form a Schiffbase. Deprotonation of this species yields a nucleophilic enamine, which condenses with electrophilic aldehydes in a second step to form a new carbon-carbon bond. Subsequent hydrolysis of the Schiffbase releases product and regenerates the active catalyst. 

The glycolytic pathway enzyme fructose-1,6-bisphosphate aldolase forms an acyl-enzyme intermediate with its ketone substrate fructose 1,6-bisphosphate. Given that the enzyme contains a lysine residue that is essential for its activity, what type of covalent intermediate is likely to be formed ... 

Like transketolase, transaldolase (TA, E.C. 2.2.1.2) is an enzyme in the oxidative pentose phosphate pathway. TA is a class one lyase that operates through a Schiff-base intermediate and catalyzes the transfer of the C(l)-C(3) aldol unit from D-sedoheptulose 7-phosphate to glyceraldehyde-3-phosphate (G3P) to produce D-Fru 6-P and D-erythrose 4-phosphate (Scheme 5.59). TA from human as well as microbial sources have been cloned.110 111 The crystal structure of the E. coliu and human112 transaldolases have been reported and its similarity to the aldolases is apparent, since it consists of an eight-stranded (o /(3)s or TIM barrel domain as is common to the aldolases. As well, the active site lysine residue that forms a Schiff base with the substrate was identified.14112 Thus, both structurally and mechanistically it is related to the type I class of aldolases. 

The development of the concept of reactive immunization yielded more effective antibody aldolases.119-120 In this new approach, rather than raise antibodies against an unreactive hapten designed to mimic the transition state, the antibodies were raised against a reactive moiety. Specifically, a p-diketone that serves as a chemical trap to imprint a lysine residue in the active site of the Ab (Scheme 5.65) was used.340 A reactive lysine is a requirement of the type I aldolase mechanism. By this method two aldolase catalytic antibodies, 38C2 and 33F12 were identified.119... 

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