Triose phosphate isomerase, equilibrium

Dihydroxyacetone phosphate (82) is a substrate for a-glycero-phosphate dehydrogenase, aldolase, and triose phosphate isomerase, and its O-alkyl ethers are intermediates in the biosynthesis of phospholipids. In neutral aqueous solution at 20 °C, dihydroxyacetone phosphate exists as an equilibrium mixture of the keto (82), gem-d o (83), and enol (84) forms, as shown by n.m.r. spectroscopy. The proportion of (82) to (83)... 

Scheme 5.—The Equilibrium Catalyzed by Triose Phosphate Isomerase.

Triose phosphate isomerase, TIM, as a nearly-diffusion-limited enzyme (Ch. 2, Section 2.2.3), catalyzes the equilibration of GAP and DHAP very efficiently. However, equilibrium concentrations of GAP were metabolized to pyruvate and further to ethanol or acetate, so the stoichiometric yield on glucose to 1,3-PPD of 42.5% was lower than the target of 50%. Thus, TIM was cloned out to prevent equilibration between the desired DHAP and the undesired side-product GAP, which successfully increased the yield beyond the minimum target... 

Triose phosphate isomerase enzyme catalyses interconversion of the 3-carbon triose phosphate dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (D-GAP). The reaction is just the transfer of the pro-R hydrogen from carbon 1 of DHAP stereospecifically to carbon 2 to form the D-isomer of GAP (Figure 2). Although the equilibrium constant on the enzyme is not known, Keq for the overall reaction is 300 to 1 in favour of DHAP. The large magnitude of this number arises from the combination of an apparent Keq of 22 vith a hydration equilibrium of 29 for the hydrated and unhydrated forms of D-GAP (Trentham ei al., 1969) only the unhydrated forms of the triose phosphates are substrates for or even bind to the isomerase (Vebb eial., 1977). 

The enzyme triose phosphate isomerase catalyzes the reaction g yceraldehyde-3-phosphate dihydroxyacetone phosphate. If the equilibrium constant is 22.0 at 25 C, calculate AG .for the reaction,... 

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).

An economically viable alternative to the synthesis of deoxyribonuclosides has been developed as a two stage process involving 2-deoxy-D-ribose 5-phosphate aldolase (DERA) (Fig. 6.5.14) (Tischer et al. 2001). The first step was the aldol addition of G3P to acetaldehyde catalyzed by DERA. G3P was generated in situ by a reverse action of EruA on L-fructose-1,6-diphosphate and triose phosphate isomerase which transformed the DHAP released into G3P. In a second stage, the action of pentose-phosphate mutase (PPM) and purine nucleoside phosphorylase (PNP), in the presence of adenine furnished the desired product. The released phosphate was consumed by sucrose phosphorylase (SP) that converts sucrose to fructose-1-phosphate, shifting the unfavorable equilibrium position of the later reaction. 

Different rates for a and 3-glucose translocation flux of carbon through most glycolysis intermediates to end products evaluated a- and 3-fructose biphosphates are in anomeric equilibrium in yeast9 but not in bacterium, aldolose-triose phosphate isomerase triangle is near equilibrium. 

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. 

An enzyme catalyzes the interconversion of dihydroxyacetone phosphate into D-phosphoglyceraldehyde in presence of NAD. Thus, triose phosphate isomerase breaks a carbon-hydrogen bond in the hydroxymethyl group of the D-phosphoglyceraldehyde to yield dihydroxyacetone phosphate. The equilibrium of that reaction favors the formation of the dihydroxyacetone phosphate. From the description of the glycolytic pathway, it is evident that dihydroxyacetone phosphate is produced in two different enzymic reactions, catalyzed by aldolase or triose phosphate isomerase. The exact mechanism of the reaction is not known, but it has ben suggested that it involves the formation of an enolate anion that is bound to the enzyme. 

CO2, present in the form of HCO3—, is fixed in the acceptor, ribulose-1,5-diphosphate, by means of the enzyme carboxydismutase. An intermediate with 6 C atoms is formed, the identity of which is still unknown. This substance is unstable. It decomposes into two molecules of 3-phosphoglyceric acid. The latter is then reduced to 3-phosphoglyceraldehyde by means of the ATP and NADPH + H+ formed in the primary processes. 3-Phosphoglyceraldehyde exists in equilibrium with its isomer, dihydroxy acetone phosphate. The equilibrium is controlled by the enzyme triose phosphate isomerase. 3-Phosphoglyceralde-hyde and dihydroxy acetone phosphate ar referred to collectively as triose phosphate. 

Because dihydroxyacetone phosphate and glyceraldehyde 3-phosphate enolize to give a common intermediate, they exist in equilibrium. The enzyme triose phosphate isomerase efficiently catalyzes the isomerization. Although the enediol intermediate is chiral, the enzyme forms only the i enantiomer of glyceraldehyde 3 phosphate. In aqueous solution, an acid-catalyzed reaction would yield a racemic mixture of aldehyde 3-phosphate. 

Trioses. n-Glyceraldehyde (formula in Section 1) is the dehydrogenation product of glycerol. More important, however, is 3-phosphoglyceraldehyde, an intermediate in the degradation of carbohydrates (Chapt. XVII-6), which is in equilibrium with dihydroxyacetone phosphate the attainment of equilibrium is catalyzed by the enzyme triose phosphate isomerase (cf. Section 7). 

By measuring the relative intensities of the fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde, it was possible to show that the aldolase reaction is in equilibrium in the cell. Similar measurements on dihydroxyacetone phosphate and glyceraldehyde-3-phosphate showed that the triose phosphate isomerase reaction is not in equilibrium. It was also found that the adenylate kinase reaction, 2ADP ATP + AMP, is in equilibrium in the intact cell without added oxygen or glucose. 

The keto-triose and the aldo-triose are in equilibrium through the enol form common to both of them 96% is in the keto-form, dihydroxyacetone phosphate. The attainment of equihbrium is accelerated by the enzyme triose-pkosphate isomerase which possesses an astoundingly high turnover number (several hundred thousand molecules per minute). Hence, the small amounts of gyceraldehyde phosphate present are replenished as soon as they are used up in the subsequent reaction. 

Glucose-6-P is then isomerized by phosphohexose isomerase to fructose-6-P (making up 30% of the equilibrium mixture). Another kinase phosphorylates the 1-position and the resulting fructose diphosphate is cleaved in an equilibrium reaction to two trioses, namely dihydroxyacetone phosphate (C-1 to C-3) and glyceral-dehyde phosphate (C-4 to C-6). The equilibrium mixture is composed of 89% hexose and 11% triose (under the conditions of Meyerhof s measurements) condensation, therefore, is the preferred (= exergonic) reaction. The reaction is analogous to the aldol condensation described in organic chemistry (Chapt. 1-2, XV-5). Catalysis of the reverse reaction by the enzyme aldolase is explained by the fact that enzymes always catalyze up to the equilibrium. ... 

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