Class I Fructose 1,6-Bisphosphate Aldolase

Fig. 2. Schematic representation of substrate binding and C-C bond formation for the class I fructose 1,6-bisphosphate aldolase from rabbit muscle...

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]. 

Hall, D. R., Leonard, G. A., Reed, C. D., Watt, C. I., Berry, A., Hunter, W. N. The Crystal Structure of Escherichia coli Class II Fructose-1,6-Bisphosphate Aldolase in Complex with Phosphoglycolohydroxamate Reveals Details of Mechanism and Specificity. J. Mol. Biol. 1999, 287, 383-394. 

The D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes in vivo the equilibrium addition of (25) to D-glyceraldehyde 3-phosphate (GA3P, (18)) to give D-fructose 1,6-bisphosphate (26) (Figure 10.14). The equilibrium constant for this reaction of 10 strongly favors synthesis [34]. The enzyme occurs ubiquitously and has been isolated from various prokaryotic and eukaryotic sources, both as class I and class II forms [30]. Typically, class I FruA enzymes are tetrameric, while the class II FruA are dimers. As a rule, the microbial class II aldolases are much more stable in solution (half-lives of several weeks to months) than their mammalian counterparts of class I (few days) [84-86]. 

N. Blom, J. Sygusch, Product binding and role of the C-termlnal region in class I D-fructose 1,6-bisphosphate aldolase, Nat. Struct. Biol. 4 (1997), 36-39... 

Class I aldolases of mammals and other vertebrates can be subdivided into three distinct isoenzymes.143,331 Identification of the parental aldolases A, B, and C has been made from their substrate specificities (ratio of activity towards D-fructose 1,6-bisphosphate and towards D-fructose 1-phosphate), electrophoretic mobilities, tissue distribution, and specific immunological properties. Aldolase A is the major form, present in muscle aldolase B, the predominant form in liver and kidney and aldolase C, present in brain with aldolase A. In tissues where more than one aldolase isoenzyme occurs, a hybrid form is often observed.331... 

Class II aldolases are normally dimeric, having a molecular weight for the subunit lying352 between 30,000 and 40,000. The Km (for D-fructose 1,6-bisphosphate) of these aldolases may be as high as 300 /xM, in contrast to 5 pM for Class I aldolases.352-354 The enzyme shows no activity toward D-fructose 1-phosphate. Class II enzymes are inhibited by EDTA and require Zn2+ or other metals for catalytic activity. The metal ion may involve polarization of the carbonyl group... 

The D-fractose 1,6-bisphosphate aldolase (FraA EC 4.1.2.13) catalyzes in vivo the glycolytic equilibrium addition of 22 to D-glyceraldehyde 3-phosphate (15) to give d-fructose 1,6-bisphosphate (23). The equilibrium constant of 10" strongly favors synthesis [43]. The class I FruA isolated from rabbit muscle has been the most extensively investigated aldolase for mechanistic, structural, and preparative purposes. Its stabiUty in solution is limiting, but the class I FruA (monomeric) from Staphylococcus carnosus [91] or the Zn -dependent, type II aldolase from E. coli [92] are much more stable with half-lives up to several weeks or months as compared to only a few days [93]. Typically, type I FruA enzymes are tetrameric proteins composed of subunits of approximately 40 kDa, while the type II FruA are dimers of approximately 39-kDa subunits. 

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. 

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