Aldolase enzyme active site

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. 

In reactions catalyzed by DHAP-aldolases, hydroxylated aldehydes are generally superior to unsubstituted aldehydes presumably because of their higher reactivity (electrophilicity), higher affinity to the enzyme active site (lower values), and the fact that the products are stabilized by the formation of cyclic isomers [42].Accordingly,substrates with dual 2- or 3-hydroxyaldehyde termini seemed to be a logical choice for potential tandem aldolizations. 

Aldolase is an example of an enzyme that uses electrophilic covalent catalysis. The amine of an active site lysine forms an imine (Section 10.5.2) with the carbonyl of fructose-1,6-bisphosphate. This more reactive imine electron sink allows a reverse aldol reaction to occur via the less basic enamine rather than the more basic enolate ion. Tautomerization of the resulting enamine to an imine, then hydrolysis, releases DHAP and returns the enzyme active site lysine to the free anime, ready for the next cycle. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

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. 

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 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). 

Figure 2.20 The two mechanisms of aldolases. Group 1 enzymes from animals and higher plants use an amino group in the enzyme to form a Schiff s base intermediate to activate the aldol donors. Group II enzymes from lower organisms, use a metal ion, usually Zn " in the active site to form an enolate intermediate. The two mechanisms are examplified by fiuctose-1,6-diphosphate aldolase, a very important aldolase in synthesis and breakdown of sugars.

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>. 

Deprotonation of 1 at C3 yields an ene-diolate intermediate to which phospho-glycolohydroxamate (PGH) 9 bears a close structural resemblance (Scheme 2.2.5.3). In collaboration with J. V. Schloss, 9 was found to be a potent inhibitor not only of FucA, but indeed also of all currently accessible Class 11 aldolases with Ki in the nanomolar range [12]. Obviously, the hydroxamate mimics very effectively an advanced catalytic intermediate or transition state that is shared by these enzymes, and seems to be bound by all Zn -dependent aldolases in a very similar fashion. Interestingly, when the active-site Zn is replaced by Co ions, catalytic activity is restored and actually becomes higher than with native zinc cofactor. However, the RhuA Co complex catalyzes an oxygenase reaction that consumes... 

II aldolases FucA and RhuA from E. coli have been crystallized solution of their spatial structures confirmed a close similarity in their overall fold [14]. Both enzymes are homotetramers in which subunits are arranged in C4 symmetry. The active site is assembled in deep clefts at the interface between adjacent subunits, and the catalytic zinc ion is tightly coordinated by three His residues. From X-ray... 

In concert, structure determinations and enzymological studies for catalytic rates and product distributions with structurally varied aldehydes of native enzymes and numerous active-site mutants have allowed us to derive a conclusive blueprint for the catalytic cycle of FucA (Fig. 2.2.5.2). The proposed mechanism, which has general implications for other metal-dependent aldolases, is able to rationalize all key stereochemical issues successfully ]15]. Independent work by other groups has recently provided further insight into related proteins with Fru A and TagA specificity [16]. 

There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2 the Zn2+ polarizes the carbonyl group... 

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,... 

Figure 13-7 Interaction of the bound zinc ion of L-fuculose-l-phosphate aldolase and catalytic side chains with the substrate in the active site of the enzyme as revealed by X-ray crystallography and modeling. See Dreyer and Schulz.193...

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. 

Redesign of an Enzyme s Active Site KDPC Aldolase... 

The carbon-carbon forming ability of aldolases has been limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has most often not produced the desired effect. Directed evolution approaches have furnished novel activities through multiple mutations of residues involved in recognition in no instance has a key catalytic residue been altered while activity is retained. Random mutagenesis resulted in a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile (Wymer, 2001). 

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). 

Redesign of an Enzyme s Active Site KDPG Aldolase 331 11.5 Comparison of Directed Evolution Techniques 331... 

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. 

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