Yeast aldolase

FIGURE 16.8 (a) Phosphoglycolohydroxamate is an analog of the enediolate transition state of the yeast aldolase reaction, (b) Purine riboside, a potent inhibitor of the calf intestinal adenosine deaminase reaction, binds to adenosine deaminase as the 1,6-hydrate. The hydrated form of purine riboside is an analog of the proposed transition state for the reaction. 

Yeast aldolase is a zinc metalloenzyme (138). The metal ion is easily dissociated and activity can be restored to the apoenzyme by Zn2+,... 

Although this review concerns those reactions catalyzed by iminium ion formation, it is important to note that there are enzymatic reactions that could logically be catalyzed by iminium ion formation but which are not. Yeast aldolase, for example, is the best known case [26] (see Ch. 6). This enzyme is metal ion dependent, does not demonstrate the loss activity in the presence of both substrate and borohydride, and is sensitive to inhibition by EDTA. The reaction catalyzed by this enzyme is identical to that catalyzed by the imine-forming enzyme, and even has evolved to exhibit the same retention stereochemistry. Another example is A -3-oxosteroid reductase which is responsible for the NADPH-dependent reduction of the enone double bond to the corresponding dihydrosteroid [124]. Even though iminium ion formation would increase the reactivity of this substrate toward the -hydride addition, a demonstrated lack of the required oxygen exchange proves that this does not occur. 

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 ... 

Another example of a activation of enolization of the substrate, dihy-droxyacetone phosphate (DHAP), occurs with yeast aldolase 22), a Zn metalloenzyme in which the Zn may be removed and replaced by Mn (23). 

FIGURE 8.6 A comparison of estimated and published cross-sections for a set of 32 peptides obtained from four different tryptic protein digests (alcohol dehydrogenase (yeast), aldolase (rabbit), creatine phosphokinase (bovine), and hemoglobin (rabbit)). For each peptide the 2+ charge state was used. The same peptides were analyzed twice with an interval of eight months between experiments. (Reproduced from Thalassinos, K. Grabenauer, M. Slade, S.E. Hilton, G.R. Bowers, M.T. Scrivens, J.H. Ana/. Chem. 2009,81, 248-254. With permission from the American Chemical Society.)... 

Aldolase then catalyses the reversible cleavage of the six-carbon molecule into two three-carbon molecules, triose phosphates. Yeast aldolase is inactivated by cysteine and may be reactivated by Zn +, Fe + or Co + ions. The triose phosphates are a mixture of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. Only the latter undergoes further change in the EMP pathway, but an equilibrium between the two is maintained by enzymic conversion of some of the dihydroxyacetone phosphate into glycer-aldehyde-3-phosphate, catalysed by the enzyme triose-phosphate isomerase. 

Muscle aldolase is strongly inhibited by traces of heavy metals and its activity is not decreased by metal binding reagents such as cysteine and a,a -dipyridyl. Yeast aldolase, which has been extensively purified by Warburg, is inactivated by cysteine and reactivated by ferrous, zinc, or cobaltous ion. Aldolase of Clostridium perfringens, on the other hand, is reactivated by ferrous or cobaltous ions in the presence of cysteine. Pea aldolase is not inhibited by heavy metals nor by cysteine and is not activated by ferrous or cobaltous ions. It is puzzling that although each of these aldolases catalyzes the same thermodynamic reaction, they still possess markedly different activation requirements. 

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... 

D-afe-o-Heptulose (sedoheptulose) (XXXVII) has been synthesized from D-erythrose (XXXVIII) plus triose phosphate, using an aldolase preparation from peas.169 Aldolases from yeast and from rat liver also form heptu-lose phosphate from these substrates.7S(o) 170(a) Crystalline muscle aldolase causes the formation of L-jrZwco-heptulose (XXXVIIa) from a mixture of L-erythrose (XXXVTIIa) and hexose diphosphate.170(b)... 

I, 7-diphosphate.170 1 (f> This tetrose phosphate is involved with phosphoenol pyruvate in the formation of shikimic acid via 3-deoxy-2-keto-D-ara6ino-heptonic acid 7-phosphate and, hence, of aromatic compounds.170(d) A synthesis of the tetrose phosphate has been described.170 1 Aldolase shows a high affinity for the heptulose diphosphate and, compared with that for D-fructose 1,6-diphosphate, the rate of reaction is about 60 %. The enzyme transaldolase, purified 400-fold from yeast, catalyzes the following reversible reaction by transfer of the dihydroxyacetonyl group.l70(o>... 

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... 

The transaldolase (EC 2.2.1.2) is an ubiquitous enzyme that is involved in the pentose phosphate pathway of carbohydrate metabolism. The class I lyase, which has been cloned from human [382] and microbial sources [383], transfers a dihydroxyacetone unit between several phosphorylated metabolites. Although yeast transaldolase is commercially available and several unphosphorylated aldehydes have been shown to be able to replace the acceptor component, preparative utilization has mostly been limited to microscale studies [384,385] because of the high enzyme costs and because of the fact that the equilibria usually are close to unity. Also, the stereochemistry of transaldolase products (e.g. 38, 40) [386] matches that of the products from the FruA-type DHAP aldolase which are more effortlessly obtained. 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

Metals are used not only as labels in histochemistry and immunochemistry, but also for studying protein structure and properties. Some metal ions can serve as valuable probes by replacing the original ions in different metaloenzymes or other metaloproteins. For instance, cobalt can replace zinc on the active side of carboxy-peptidase, aldolase, carbonic anhydrase, phosphatase or yeast alcohol dehydrogena. ... 

Heinisch, J.J., Rodido, R. (1996) Fructose-1,6 biphospohate aldolase, triose phosphate iso-merase, glyceraldelnde-3-phospkate deshidrogenases and phosphogjycerate mutase. In F.K. Zimmerman K.D. Entian (Eds.), Yeast Sugar Metabolism Biochemistry, Genetics, Biotechnology, and Applications (pp. 119-140). Boca Raton CRC Press. 

---