Isocitrate, determination

Figure 4. The citrate cycle. There is complete oxidation of one molecule of acetyl-CoA for each turn of the cycle CH3COSC0A + 2O2 - 2CO2 + H2O + CoASH. The rate of the citrate cycle is determined by many factors including the ADP/ATP ratio, NAD7NADH ratio, and substrate concentrations. During muscle contraction, Ca is released from cellular stores (mainly the sarcoplasmic reticulum) and then taken up in part by the mitochondria (see Table 2). Ca " activates 2-oxoglutarate and isocitrate dehydrogenases (Brown, 1992). Succinate dehydrogenase may be effectively irreversible. Enzymes ...

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 overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and a-lcetoglutarate dehydrogenase. These fluxes are largely determined by the concentrations of substrates and products the end products ATP and NADH are inhibitory, and the substrates NAD+ and ADP are stimulatory. 

NOTE The standard solutions listed above are for the most common acids found in several common juices. These are not all the acids found in each juice. For example, isocitric and citric acid are commonly found in raspberry juice, and the ratio of the two is an important factor for determining adulteration of the juice. 

In the analysis of fruit juices, it is important to determine the levels of the individual acids to assess authenticity and quality. A range of these acids can be determined using an enzyme-linked assays and these procedures have been collaboratively tested and published in the IFU compendium of methods (citric no. 22, isocitric no. 54, D-malic no. 64, L-malic no. 21 and D-and L-lactic acids no. 53). r-Biopharm now distributes the Boehringer Mannheim kits to assess the levels of these acids. Similar kits are available from other suppliers. 

Figure 6 Schematic diagram of the active site of the E. coli NADP-IDH with bound 2A,3>S-isocitrate (A) and the active site of T. thermophilus NAD-IMDH with bound 2R,3S-isopropylmalate (B). A prime indicates the residues donated from the second subunit, a, 3 refers to a- and P-carboxyl groups, respectively. The major determinants of substrate specificity are residues Seri 13 and Asnll5 in the NADP-IDH, and Glu87 and Leu90 in the NAD-IMDH, which interact with the unique y-moieties of the substrates. (Modified from Ref. 15.)...

AM Dean, AK Shiau, DE Koshland Jr. Determinants of performance in the isocitrate dehydrogenase of Escherichia coli. Protein Sci 5 341-347, 1995. 

JH Hurley, RD Chen, AM Dean. Determinants of cofactor specificity in isocitrate dehydrogenase structure of an engineered NADP —> NAD specificity-reversal mutant. Biochemistry 35 5670-5678, 1996. 

The NADP-IDH from Escherichia coli has been thoroughly studied. It is a dimeric protein of two identical 40-kDa subunits. High-resolution X-ray crystal structures have been determined for the enzyme with and without substrate [16,17], and for the pseudo-Michaelis complex of the enzyme with isocitrate and NADP [18], Structures of sequential intermediates formed during the catalytic action of IDH are also available [19], Additionally, the kinetic and catalytic mechanisms have been determined in detail [20], Amino acid residues which are involved in interactions with substrate, coenzyme, metal ions, and catalysis have been identified [10,21],... 

In the present experiment, you will determine the activity of isocitrate dehydrogenase extracted from pork heart muscle. The commercial preparation comes in powder form and it uses NADP+ rather than NAD+ as a coenzyme. The basis of the measurement of the enzyme activity is the absorption spectrum of NADPH. This reduced coenzyme has an absorption maximum at 340 nm. Therefore, an increase in the absorbance at 340 nm indicates an increase in NADPH concentration, hence the progress of the reaction. We define the unit of isocitrate dehydrogenase activity as one that causes an increase of 0.01 absorbance per min. at 340 nm. 

Partitioning between the Citric Acid and Glyoxylate Cycles In an organism (such as E. coll) that has both the citric acid cycle and the glyoxylate cycle, what determines which of these pathways isocitrate will enter ... 

Hydroxylase activity was determined in a reaction mixture containing mitochondrial protein and 11-deoxycorticosterone (60 /xAf). The reaction was started by the addition of 10 mAf isocitrate as the source of reducing equivalents. At intervals during the incubation, samples were removed... 

Fluorimetric methods involving six dehydrogenases have been used to determine the concentrations of 21 of the more common organic acids (58). Sensitivity of the methods range from 0.02 fxg/m for determination of D-isocitric acid with isocitrate dehydrogenase to 200 /xg/ml for determination of D-tartaric acid with malate dehydrogenase. 

Glusker, J. P., Patterson, A. L., Love, W. E., and Dornberg, M. L. X-ray crystal analysis of the substrates of aconitase. IV. The configuration of the naturally occurring isocitric acid as determined from potassium and rubidium salts of its lactone. Acta Cryst. 16, 1102-1107 (1963). 

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