Muscle enzymes aldolase

Of the several aldolases that are now commercially available, the rabbit muscle enzyme with a useful specific activity of 20 Umg-1 still remains the most cost-efficient catalyst for preparative work, although the reported higher stability of the Staphylococcus carnosus enzyme is a challenge [252,285], Literally hundreds of aldehydes have so far been tested successfully by enzymatic... 

The copious muscle enzyme efflux in Duchenne-type muscular dystrophy, giving gross serum elevations despite the rapid serum clearance, may well deplete some muscle enzymes that so much aldolase still remains may indicate a replacement so rapid that, if applied to the transaminases and to lactic dehydrogenase, the muscle content may be maintained or even increased, since their serum elevations, though considerable, are proportionately much less than that of aldolase. 

Generalized muscle rigidity (found in 70% of the patients involved) and a progressive rise in body temperature (sometimes beyond 43°C) are the main clinical features, often associated with tachycardia, hypoxia, metabolic acidosis, cardiac dysrhythmias and, less often, disseminated intravascular coagulation, cerebral edema, and acute renal insufficiency. Diagnosis relies on the clinical signs, that is muscle rigidity and hyperpyrexia, and on raised serum activities of skeletal and cardiac muscle enzymes, for example aldolase and creatine kinase. 

A further example are Laser Raman spectroscopy resonance studies of the enzyme aldolase catalyzing a key reaction in the muscle cells. Zerbi et al.71) investigated resonance Raman spectra of labelled aldolase with argon ion and krypton ion lasers. 

Fructose 1-phosphate is further metabolized to dihydroxyacetone phosphate (DHAP) and glyceraldehyde by the hepatic isoform of the enzyme aldolase, which catalyzes a reversible aldol condensation reaction. Aldolase is present in three different isoforms. Aldolase A is present in greatest concentrations in the skeletal muscle, whereas the B isoform predominates in the liver, kidney, and intestine. Aldolase C is the brain isoform. Aldolase B has similar activity for either fructose 1,6-bisphosphate (F16BP) or FIP however, the A or C isoforms are only slightly active when FIP is the substrate. 

An exhaustive review of all of the types of reactions that are catalyzed by metal-requiring enzymes and the specific functions of these metals, as currently understood, is beyond the scope of this chapter. To complicate this general area of investigation, even within a single group of enzymes, the metal ions may play different roles in the catalytic processes for reaction-related enzymes. Not all enzymes of a specific class necessarily require a cation for activity. In some cases, the roles of the cation may be substituted by specific amino acid residues in the protein. A classic example of such a case is the muscle and yeast fmctose-bisphosphate aldolases. The muscle enzyme catalyzes the aldol condensation using a Schiff base intermediate to activate the substrate, whereas the yeast enzyme is a Zn +-metalloenzyme (1). The cation appears to serve as the electrophile in the activation of the substrate for the same reaction. 

The observation of net retention of configuration by the muscle enzyme, as well as the other aldolases, suggests that a single active site base could alternatively function in a general base and general acid capacity in the exchange and condensation reactions, respectively (Fig. 7) (76). However, the stereochemistry does not absolutely require a single-base mechanism. For example, Hupe and co-workers propose a two-base mechanism in which the phosphate at C-1 of the... 

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 lysiae S-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily ia microorganisms and utilize a divalent ziac to activate the electrophilic component of the reaction. The most studied aldolases are fmctose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn " -containing aldolase from E. coli. In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetone phosphate [57-04-5] (DHAP). 

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. 

The class I FruA isolated from rabbit muscle aldolase (RAMA) is the aldolase employed for preparative synthesis in the widest sense, owing to its commercial availability and useful specific activity of 20 U mg . Its operative stability in solution is limiting, but the more robust homologous enzyme from Staphylococcus carnosus has been cloned for overexpression [87], which offers unusual stability for synthetic purposes. Recently, it was shown that less polar substrates may be converted as highly concentrated water-in-oil emulsions [88]. 

Dihydroxyacetone phosphate reacts with D-glycerose in the presence of aldolases of muscle and liver to give D-fructose 1-phosphate (XII) exclusively, whilst DL-glycerose forms equimolar proportions of D-fructose 1-phosphate (XII) and L-sorbose 1-phosphate (XIII).65 Specificity of the enzyme is interesting in the light of Fischer and Baer s observations66 in... 

These enzymes have been found in all plant and animal tissues examined and are absent only from a few specialized bacteria. Three closely related isoenzymes are found in vertebrates.185 186 The much studied rabbit muscle aldolase A is a 158-kDa protein tetramer of identical peptide chains.186 187 Aldolase B, which is lacking in hereditary fructose intolerance, predominates in liver and isoenzyme C in brain.185... 

Representatives of all kinds have been explored for synthetic applications while mechanistic investigations were mainly focussed on the distinct FruA enzymes isolated from rabbit muscle [196] and yeast [197,198]. For mechanistic reasons, all DHAP aldolases appear to be highly specific for the donor component DHAP [199], and only a few isosteric replacements of the ester oxygen for sulfur (46), nitrogen (47), or methylene carbon (48) were found to be tolerable in preparative experiments (Fig. 7) [200,201], Earlier assay results [202] that had indicated activity also for a racemic methyl-branched DHAP analog 53 are now considered to be artefactual [203]. Dihydroxyacetone sulfate 50 has been shown to be covalently bound via Schiff base formation, but apparently no a-deprotonation occurred as neither H/D-exchange nor C-C... 

Fructose 1,6-biphosphate aldolase from rabbit muscle in nature reversibly catalyzes the addition of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate. The tolerance of this DHAP-dependent enzyme towards various aldehyde acceptors made it a versatile tool in the synthesis of monosaccharides and sugar analogs [188], but also of alkaloids [189] and other natural products. For example, the enzyme-mediated aldol reaction of DHAP and an aldehyde is a key step in the total synthesis of the microbial elicitor (—)-syringolide 2 (Fig. 35a) [190]. 

For the described approach, it is important to note that aldolases of different origin were tested and that, in contrast to L-rhamnulose-1-phosphate aldolase (RhuA), the D-fructose-1,6-biphosphate aldolase from rabbit muscle and L-fucu-lose-1-phosphate aldolase from E. coli were not active for DHAP/(R)-N- and (S)-iV-Cbz-prolinal condensation. Since RhuA accepts both, (S)-N- and (R)-N-Cbz prolinals, the chemo-enzymatic synthesis of both, hyacinthacines A and A2 isomers could be achieved. In conclusion, the origin and the particular enzyme itself... 

Carbon-carbon bond-forming reactions are some of the most important transformations in organic chemistry. Sobolov et al. [33] reported that CLCs of fructose 1,6-diphosphate aldolase from rabbit muscle are much more stable than the soluble enzyme. The synthetic potential of these CLCs was demonstrated by the preparation of a series of compounds shown in Fig. 10. 

In brain tissues, specific isoforms of glycolytic enzymes are also expressed there are specific brain isoforms for PFK (PFK-C), fructose-1,6-bisphosphate aldolase (aldolase C), enolase (enolase y), but not for GAPDH. The isoforms bear the same catalytic functions however, they could be specialized to form different ultrastructural entities. For example, muscle PFK (a dissociable tetrameric form) binds to microtubules and bundle them [94, 95], however, the brain isoenzyme (stable tetramer) does not [96]. 

Furthermore, the catalytic efficiency (K t/KM) of 84G3 for this substrate, 3.3 X 105 s-1M-1, is comparable with the efficiency of natural muscle aldolase, 4.9 X 104 s 1M 1, in the retro-aldolization of its substrate fructose 1, 6-bisphosphate (Morris and Tolan, 1994). However, these two enzymes use different substrates, and the rates were recorded at different temperatures (22°C for 84G3, 4°C for the natural aldolase). Despite this, we believe that it will be possible to develop a catalytic antibody that, under identical conditions, has a faster cat and lower KM than a natural enzyme for the same substrate. 

---