Isodtrate

The realisation that yeasts would produce dtric acid from n-paraffins was veiy attractive in the late 1960 s. Petroleum byproducts were plentiful and very cheap and there was detailed knowledge available on these processes because the use of hydrocarbon-utilising yeasts for single cell protein was well developed. The strategy was to use n-alkane to produce high yields erf dtric add-producing Candida spp. and to harvest two useful end products rather than just one. The process has not been commerdally successful however. Candida spp. produce mixtures of dtric add and isodtric add and the latter is not a useful product. In addition, since 1973 when petroleum prices rose sharply and have in fact continued to rise, the n-paraffins are no longer a cheap substrate. 

The diagram looks very promising in terms of citric acid formation in that a-oxoglutarate dehydrogenase is inactive, isodtrate dehydrogenase has veiy low activity and aconitase equilibrates 90% towards dtric add. 

Word list isodtrate, constitutive, ammonium, ATP, manganese, dtrate. 

Figure 9.2 Summary of reactions of the Krebs cycle. The names of the enzymes are dtrate synthase, aconitase, isodtrate dehydrogenase (there are two enzymes, one ubTizes NAD as the cofactor, the other NADPT it is assumed that the NAD -specific enzyme is that involved in the cycle), oxoglutarate dehydrogenase, sucdnyl CoA synthetase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase.

Gawron et al. (13,14) determined the stereochemistry of natural isocitric acid by chemical means. The results require the rrons-addition of water across the cis-aconitate intermediate double bond to produce either citrate or 2R,3S-isodtrate. Mass and NMR analyses of isotopically labeled citrate and isocitrate in the early 1960 s (15-17), defined the stercospedficities of the dehydration steps. These results led Gawron to propose the binding of cis-aconitate to the active site in two orientations differing by a 180° rotation about the double bond, as shown in Equation 2. This allows for the protonation by a base (-BH) and hydroxylation of the double bond to occur on aconitase at single, separate loci for the formation of either citrate or isocitrate. 

The most important factor in the regulation of the cycle is the NADH/NAD ratio. In addition to pyruvate dehydrogenase (PDH) and oxoglu-tarate dehydrogenase (ODH see p. 134), citrate synthase and isodtrate dehydrogenase are also inhibited by NAD deficiency or an excess of NADH+HT With the exception of isocitrate dehydrogenase, these enzymes are also subject to product inhibition by acetyl-CoA, suc-cinyl-CoA, or citrate. 

Interconversion processes (see p. 120) also play an important role. They are shown here in detail using the example of the PDH complex (see p. 134). The inactivating protein kinase [la] is inhibited by the substrate pyruvate and is activated by the products acetyl-CoA and NADH+H. The protein phosphatase [Ibj—like isodtrate dehydrogenase [3] and the ODH complex [4j-is activated by Ca. This is particularly important during muscle contraction, when large amounts of ATP are needed. Insulin also activates the PDH complex (through inhibition of phosphorylation) and thereby promotes the breakdown of glucose and its conversion into fatty acids. 

Fig. 2.13. Substrate binding site of isocitrate dehydrogenase E. coli). The interactions involved in the binding of Mg Msodtrate at the active site of isodtrate dehydrogenase are shown. After Hnrley et al., (1990), with permission.

Figure 8 (a) Extended and (b) rotated conformations of citric add and (c) extended conformation of (-l-)-isodtric acid... 

T. Yaoi, K. Miyazaki, T. Oshima, Y. Komukai, and M. Go, Conversion of the coenzyme specificity of isodtrate dehydrogenase by module replacement, J. Biochem. (Tokyo) 1996, 119, 1014-1018. 

Step 2 of Figure 29.12 Isomerization Citrate, a prochiral tertiary alcohol, is next converted into Its isomer, (2i, 35)-isodtrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an ElcB dehydration of a fiJ-hydroxy acid to give ds-aconitate, the same sort of reaction that occurs in step 9 of glycolysis fFigure 29.7). The. second step is a conjugate nucleophilic addition of water to the C=C ht>nd (Section 19.13). The dehydration of citrate takes place specifically on the pro-R artn—the one derived from oxaloacetate—rather than on the pro-S arm derived from acetyl CoA. 

Aconitase catalyzes the isomerization of citrate to isodtrate, isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate, and a-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of a-keto-glutarate to succinyl-CoA. Succinyl-CoA and the remaining intermediates are the 4-carbon intermediates of the Krebs cycle. Succinyl thiokinase catalyzes the release of coenzyme A from succinyl-CoA and the production of GTP. Succinate dehydro-... 

FIGURE 4.58 The Krebs cycle — names of intermediates. The intermediates in the cycle are citrate, isodtrate, a-ketoglutarate, succinyl-CoA, sucdnate, fumarate, malate, and oxaloacetate. Acetyl-CoA is used to introduce the acetyl group to the Krebs cycle. Carbon dioxide is its final product. 

Phosphorylation in bacteria. A bacterial enzyme whose activity is controlled by phosphorylation is isodtrate dehydrogenase. Transfer of a phospho group to the -OF4 of Ser 113 completely inactivates the... 

Oxidative decarboxylations. Isocitrate, a secondary alcohol, is oxidized by NAD in step 3 to give a ketone, which loses COj to give -ketoglutarate. Catalyzed by the enzyme isodtrate dehydrogenase, the decarboxylation is a typical reaction of a /3-keto acid, just like that in the acetoacetic ester synthesis (Section 22.8). 

STEP > Isodtrate is oxidized and loses CO i to yield o-ketoglutarate. 

Fig. 3-24. Gradient elution of inorganic and organic anions on IonPac AS5A. - Eluent (A) 0.00075 mol/L NaOH, (B) 0.1 mol/L NaOH gradient 100% A isocratically for 5 min, then linearly to 30% B in 15 min, then linearly to 86% B in 15 min flow rate 1 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations 1.5 ppm fluoride (1), 10 ppm a-hydroxybutyrate (2), acetate (3), glycolate (4), butyrate (5), gluconate (6), a-hydroxyvalerate (7), 5 ppm formate (8), 10 ppm valerate (9), pyruvate (10), monochloroacetate (11), bromate (12), 3 ppm chloride (13), 10 ppm galacturonate (14), 5 ppm nitrite (15), 10 ppm glucoronate (16), dichloroacet-ate (17), trifluoroacetate (18), phosphite (19), selenite (20), bromide (21), nitrate (22), sulfate (23), oxalate (24), selenate (25), a-ketoglutarate (26), fumarate (27), phthalate (28), oxalacetate (29), phosphate (30), arsenate (31), chromate (32), citrate (33), isodtrate (34), eis-aconitate (35), and frons-aconitate (36).

Fig. 4-10. Separation of organic acids on IonPac ICE-AS5. - Eluent 0.0016 mol/L perfluorobu-tyric acid flow rate 0.3 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations fully dissociated compounds (1), 10 ppm oxalic acid (2), 25 ppm pyruvic acid (3), and tartaric acid (4), 30 ppm malonic acid (5), lactic acid (6), malic acid (7), and acetic acid (8), 20 ppm isodtric acid (9), 30 ppm citric acid (10), 40 ppm / -hydroxybutyric acid (11), succinic acid (12), and propionic acid (13).

---