Triosephosphates

P. Derreumaux and T. Schlick. The loop opening/closing motion of the enzyme triosephosphate isomerase. Biophys. J., 74 72-81, 1998. 

Noble M E M, R K Wierenga, A-M Lambeir, F R Opperdoes, W H Thunnissen, K H Kalk, H Groendijk and W G J Hoi 1991. The Adaptability of the Active Site of Trypanosomal Triosephosphate Isomerase as Observed in the Crystal Structures of Three Different Complexes. Proteins Structure, Function and Genetics 10 50-69. 

The chemical reaction catalyzed by triosephosphate isomerase (TIM) was the first application of the QM-MM method in CHARMM to the smdy of enzyme catalysis [26]. The study calculated an energy pathway for the reaction in the enzyme and decomposed the energetics into specific contributions from each of the residues of the enzyme. TIM catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) as part of the glycolytic pathway. Extensive experimental studies have been performed on TIM, and it has been proposed that Glu-165 acts as a base for deprotonation of DHAP and that His-95 acts as an acid to protonate the carbonyl oxygen of DHAP, forming an enediolate (see Fig. 3) [58]. 

Figure 3 A possible mechanism for the isomerization of dihydroxyacetone phosphate (DHAP) to D glyceraldehyde 3 phosphate (GAP) by the enzyme triosephosphate isomerase (TIM). The general acid (Glu 165) and general base (His 95) are shown.

Citrate synthase catalyzes the metabolically important formation of citrate from ace-tyl-CoA and oxaloacetate [68]. Asp-375 (numbering for pig CS) has been shown to be the base for the rate-limiting deprotonation of acetyl-CoA (Fig. 5) [69]. An intennediate (which subsequently attacks the second substrate, oxaloacetate) is believed to be formed in this step the intermediate is thought to be stabilized by a hydrogen bond with His-274. It is uncertain from the experimental data whether this intermediate is the enolate or enol of acetyl-CoA related questions arise in several similar enzymatic reactions such as that catalyzed by triosephosphate isomerase. From the relative pK values of Asp-375... 

Figure 2.10 Examples of schematic diagrams of the type pioneered by Jane Richardson. Diagram (a) illustrates the structure of myoglobin in the same orientation as the computer-drawn diagrams of Figures 2.9b-d. Diagram (b), which is adapted from J. Richardson, illustrates the structure of the enzyme triosephosphate isomerase, determined to 2.5 A resolution in the laboratory of David Phillips, Oxford University. Such diagrams can easily be obtained from databases of protein structures, such as PDB, SCOP or CATH, available on the World Wide Web.

This motif is called a beta-alpha-beta motif (Figure 2.17) and is found as part of almost every protein structure that has a parallel p sheet. For example, the molecule shown in Figure 2.10b, triosephosphate isomerase, is entirely built up by repeated combinations of this motif, where two successive motifs share one p strand. Alternatively, it can be regarded as being built up from four consecutive p-a-p-a motifs. 

Table 4.1 The amino acid residues of the eight parallel p strands in the barrel structure of the enzyme triosephosphate isomerase from chicken muscle...

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

B35. Brown, J. R., Daar, I. O., Krug, J. R., and Maquart, L. E., Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family. Mol. Cell. Biol. 5,1694-1707 (1985). 

Dl. Daar, I. O., Artymiuk, P. J., Phillips, D. C and Maquat, L. E., Human triosephosphate iso-merase deficiency. A single amino acid substitution results in a thermolabile enzyme. Proc. Natl. Acad. Sci. U.S.A. 83,7903-7907 (1986). 

M10. Maquat, L. E Chilcote, R., and Ryan, P. M., Human triosephosphate isomerase cDNA and protein structure Studies of triosephosphate isomerase in man. J. Biol. Chem. 260, 3748-3753 (1985). 

Schneider, A.,Westwood, B., Yim, C Prchal, J., Berkow, R Labotka, R., Warner, R., and Beut-ler, E Triosephosphate isomerase deficiency Occurrence of point mutation in amino acid 104 in multiple apparently unrelated families. Am. J. Hematol. 50,263-268 (1995). 

W3. Watanabe, M., Zingg, B. C and Mohrenweiser, H. W., Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate isomerase locus. Am. J. Hum. Genet. 58, 308-316(1996). 

A major problem in unfolding studies of large proteins is irreversibility. In a study of elastase temperature-induced denaturation, second-derivative FTIR show a distinct loss of several sharp amide V features (dominant /3-sheet components and growth in broadened bands at 1645 and 1668 cm-1 (Byler et al., 2000). These features persisted on cooling, indicating lack of reversibility, a feature common to longer multidomain proteins. A graphic example of this is seen in the triosephosphate... 

Fig. 15. Thermal denaturation of triosephosphate isomerase with FTIR (upper left), second-derivative FTIR (upper right), and VCD (bottom) showing irreversible aggregation effects. The IR shift from a simple maximum at 1650-1640 cm-1 to a lower frequency distorted to low wavenumber is seen to be irreversible when the original spectrum is not recovered. The second-derivative result makes the changes more dramatic and shows the original native state spectrum to be more complex (negative second derivatives correspond to peak positions). Loss of structure is even more evident in the VCD, which loses most of its intensity at 60°C.

In further studies, Amstein and Bentley5 demonstrated the presence of aldolase and triosephosphate isomerase in fungi producing kojic acid. They also found that both production and destruction of kojic acid were rapid in media with high phosphate levels, and slow at lower phosphate levels. They preferred to consider kojic acid as a normal metabolite of the fungi, rather than as an end product. 

Figure 5. A minimal model of glycolysis One unit of glucose (G) is converted into two units of pyruvate (P), generating a net yield of 2 units of ATP for each unit of glucose. Gx, Px, and Glx are considered external and are not included into the stoichiometric matrix. A A graphical depiction of the network. B The stoichiometric matrix. Rows correspond to metabolites, columns correspond to reactions. C A list of individual reactions. D The corresponding system of differential equations. Abbreviations G, glucose (Glc) TP, triosephosphate, P, pyruvate.

It can be straightforwardly verified that indeed NK = 0. Each feasible steady-state flux v° can thus be decomposed into the contributions of two linearly independent column vectors, corresponding to either net ATP production (k ) or a branching flux at the level of triosephosphates (k2). See Fig. 5 for a comparison. An additional analysis of the nullspace in the context of large-scale reaction networks is given in Section V. 

The first reaction vi (Gx. ATP) describes the upper part of glycolysis, converting one (external) molecule of glucose (Gx) into two molecules of triosephosphate (TP), using two molecules of ATP. The second reaction v2 (TP, ADP) describes the synthesis of two molecules ATP from each molecule of TP. The third reaction v3 (ATP) describes a (lumped) overall ATP utilization. To obtain a minimal kinetic model for the glycolytic pathway, we adopt rate function similar to [96], using... 

Figure 30. A medium complexity model of yeast glycolysis [342], The model consists of nine metabolites and nine reactions. The main regulatory step is the phosphofructokinase (PFK), combined with the hexokinase (HK) reaction into a single reaction vi. As in the minimal model, we only consider the inhibition by its substrate ATP, although PFK is known to have several effectors. External glucose (Glc ) and ethanol (EtOH) are assumed to be constant. Additional abbreviations Glucose (Glc), fructose 1,6 biphosphate (FBP), pool of triosephosphates (TP), 1,3 biphosphogly cerate (BPG), and the pool of pyruvate and acetaldehyde (Pyr).

As one of its characteristic features, the Calvin cycle leads to a net synthesis of its intermediates with significant implications for the stability of the cycle. Obviously, the balance between withdrawal of triosephosphates (TP) for biosynthesis and triosephosphates that are required for the recovery of the cycle is crucial. The overall reaction of the Calvin cycle is... 

Figure 36. The Calvin cycle leads to an autocatalytic net synthesis of cycle intermediates. Upon three cycles, one triosephosphate is synthesized for export. The figure is inspired by a depiction of the Calvin cycle given on http //sandwalk.blogspot.com/2007/07/Calvin cycle regeneration.html.

The construction of the structural kinetic model proceeds as described in Section VIII.E. Note that in contrast to previous work [84], no simplifying assumptions were used the model is a full implementation of the model described in Refs. [113, 331]. The model consists of m = 18 metabolites and r = 20 reactions. The rank of the stoichiometric matrix is rank (N) = 16, owing to the conservation of ATP and total inorganic phosphate. The steady-state flux distribution is fully characterized by four parameters, chosen to be triosephosphate export reactions and starch synthesis. Following the models of Petterson and Ryde-Petterson [113] and Poolman et al. [124, 125, 331], 11 of the 20 reactions were modeled as rapid equilibrium reactions assuming bilinear mass-action kinetics (see Table VIII) and saturation parameters O1 1. 

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