Triose phosphate isomerase assay

As depicted in Figure 6.8 the stability screening was based on DERA activity assay, the retro-aldol reaction of 2-deoxy-D-ribose 5-phosphate to acetaldehyde and D-glyceraldehyde 3-phosphate. D-glyceraldehyde 3-phosphate is further converted by the auxiliary enzymes triose phosphate isomerase and glycerol phosphate dehydrogenase. As the latter reaction consumes NADH it can be measured spectro-pho to metrically by the decrease in absorbance at 340 nm. 

Fig. 17. Nucleotidyltransferase substrate screening assay (NUSSA). A Nucleotide sugar-synthesizing nucleotidyltransferase (EC 2.7.7), B PPrdependent phosphofructokinase (EC 2.7.2.90), C Aldolase (EC 4.1.2.13), D Triose-phosphate isomerase (EC 5.3.1.1), E Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) [336]...

Figure 8.17 Enzyme coupled assay. Triose Phosphate Isomerase (TIM) assay system with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) colourimetric coupled assay for detection purposes. In this version of the TIM assay, dihydroxyacetone phosphate (DHAP) is converted enzymically to glyceraldehyde 3-phosphate (GAP) that is onward converted to glycerol phosphate (GP) by means of the coupled enzyme GAPDH enzyme that uses the reverse-colourimetric reductant NADH.

Figure 8.58 Schematic illustration of reaction coordinate diagram of Triose Phosphate Isomerase (TIM) enzyme illustrating near perfect energy landscape pathway allowing for near perfect 1 1 1 stoichiometric equilibrium between all enzyme-bound species optimal for flux through from one enzyme-bound species to another. Enzyme turnover rate kobs is at the diffusion limit, the rate determining step is the association of dihydroxy acetone phosphate (DHAP) with the TIM catalytic site, see Fig. 8.1, hence chemistry is not rate limiting. Therefore, TIM is considered a perfect enzyme For TIM enzyme assay see Fig. 8.17 for TIM enzyme mechanism see Fig. 8.49 (illustration adapted from Knowles, 1991, Fig. 2).

This enzyme catalyses one of the intermediate steps of glycolysis. The reaction is not considered to be a rate-lunitmg step, but it may be used as an indicator reaction for the estimation of phosphofructokinase activity which is thought to be rate limiting. Serum levels of aldolase are elevated in diseases of skeletal muscle and in the presence of tumours in many tissues. Aldolase may be assayed by an ultraviolet method using triose phosphate isomerase as an auxiliary reaction and glycerol-3-phosphate dehydrogenase as an indicator reaction [219]. The trioses can be trapped as formed with the aid of... 

Scheme 15.3 TK assays based on cascade reactions. ADH, alcohol dehydrogenase TPI, triose phosphate isomerase BSA, bovine serum albumin PPO, polyphenoloxidase.

Spectrophotometric assay (BIO, L7, S29) is accomplished by a similar incubation, without hydrazine, where excess added triosephosphate isomerase converts triose phosphate formed to DAP, in turn removed as glycerophosphate by reduced nicotinamide-adenine dinucleotide (NADH2, formerly termed DPNH) and added glycerophosphate dehydrogenase. 

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