Isomerase triosephosphate

Another type of disorder-to-order transition is the so-called loop-cap transition in certain enzymes.279,280 This structural change is associated with a highly mobile loop of residues, from 6 to 12 amino acid residues in length, that are located near the active site. Such a structural feature has been identified in the enzyme triose phosphate isomerase (TIM). TIM makes an especially interesting case study for theoretical analysis of the loop-cap transition because there exist X-ray,279,313 kinetic,121 and thermodynamic data121 for this enzyme. 

It is likely that the entropy loss in closing the loop partially offsets the en-thalpic gain on binding. In this way the enzyme can be highly specific, yet not bind the substrate too strongly. Further, it is possible that the disorder-to-order transition in the formation of the enzyme substrate complex raises its 

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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). 

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). 

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. 

Parallel /3 structure usually forms large, moderately twisted sheets such as in Fig. 23, although occasionally it rolls up into a cylinder with helices around the outside (e.g., triosephosphate isomerase). Large antiparallel sheets, on the other hand, usually roll up either partially (as in the first domain of thermolysin or in ribonuclease) or completely around to join edges into a cylinder or barrel. Occurrence, topology, and classification of /3 barrels will be discussed in Section III,D, but here we will consider the interaction between the /3 sheets on opposite sides of the barrel, especially in terms of the angle at which opposite strands cross. 

Fig. 29. An assortment of/3 barrels, viewed down the barrel axis (a) staphylococcal nuclease, 5-stranded (b) soybean trypsin inhibitor, 6-stranded (c) chymotrypsin, 6-stranded (d) immunoglobulin (McPC603 CH1) constant domain, 7-stranded (e) Cu,Zn superoxide dismutase, 8-stranded (f) triosephosphate isomerase, 8-stranded (g) im-... 

Fig. 71. Examples of protein domains with different numbers of layers of backbone structure (a) two-layer cytochrome c (b) three-layer phosphoglycerate kinase domain 2 (c) four-layer triosephosphate isomerase. The arrows above each drawing point to the backbone layers.

A. Singly wound parallel /3 barrels Triosephosphate isomerase Pyruvate kinase domain 1 KDPG aldolase ( )... 

Fic. 90. Triosephosphate isomerase as an example of a singly wound parallel /3 barrel, (a) a-Carbon stereo, viewed from one end of the barrel (b) backbone schematic, viewed as in a (c) a-carbon stereo, viewed from the side of the barrel (d) backbone schematic, viewed as in c (e) topology diagram showing the + lx right-handed connections between the fi strands. 

The ultimate objective of an X-ray cryoenzymological study is the mapping of the structures of all kinetically significant species along the reaction pathway. In the case of ribonuclease A this has been largely achieved, as described above. Other enzymatic reactions now await application of the same techniques. Unfortunately, not all crystalline enzymes lend themselves to study by this method. In some cases it may be impossible to find a suitable cryoprotective mother liquor in others, the reaction may occur too rapidly at ordinary temperature. A reaction with Acat of 10 seconds and an activation enthalpy of —6 kcal mol will not be quenched even at — 75°C. The approach we have described in this article can be applied to only a small number of enzymes. Two likely candidates for successors to ribonuclease are the enzymes yeast triosephosphate isomerase and porcine pancreatic elastase. 

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