Of triose phosphate

R. C. Wade, M. E. Davis, B. A. Luty, J. D. Madura, and J. A. McCammon. Gating of the active site of triose phosphate isomerase Brownian dynamics simulations of flexible peptide loops in the enzyme. Biophys. J., 64 9-15, 1993. 

Another important parallel /3-array is the eight-stranded parallel j8-barrel, exemplified in the structures of triose phosphate isomerase and pyruvate kinase (Figure 6.30). Each /3-strand in the barrel is flanked by an antiparallel a-helix. The a-helices thus form a larger cylinder of parallel helices concentric with the /3-barrel. Both cylinders thus formed have a right-handed twist. Another parallel /3-structure consists of an internal twisted wall of parallel or mixed /3-sheet protected on both sides by helices or other substructures. This structure is called the doubly wound parallel j8-sbeet because the structure can be... 

FIGURE 6.30 Parallel /3-array proteins—the eight-stranded /3-barrels of triose phosphate iso-merase (a, side view, and b, top view) and (c) pyruvate kinase. (Jane Richardson)... 

Knowles, J., and Albery, W., 1977. Perfection in enzyme catalysis The energetics of triose phosphate isomerase. Accounts of Chemical Research 10 105-111. 

TRIACYLGLYCEROLS PHOSPHOGLYCEROLS ARE FORMED BY ACYLATION OF TRIOSE PHOSPHATES... 

Figure 14. Principle for measuring bidirectional fluxes by 13C metabolic flux analysis. In a carbon labeling experiment, 1 13C glucose is provided in the medium, and the culture is grown until a steady state is reached. Glucose can either go directly via the hexose phosphate pool (Glu 6P and Fru 6P) into starch, resulting in labeling hexose units of starch only at the Cj position, or it can be cleaved to triose phosphates (DHAP and GAP), from which hexose phosphates can be resynthesized, which will result in 50% labeling at both the Ci and the C6 position (assuming equilibration of label by scrambling at the level of triose phosphates). From the label in the hexose units of starch, the steady state fluxes at the hexose phosphate branchpoint can be calculated for example, if we observe 75% label at the Ci and 25% at the C6 position, the ratio of vs to V7 must have been 1 to 1. All other fluxes can be derived if two of the fluxes of Vi, V6, and V7 are known (e.g., V2 vi V3 V5 + v6).

The example of triose phosphate isomerase in Box 13.6 provides us with an easily understood analogy. 

A quantitative expression developed by Albery and Knowles to describe the effectiveness of a catalyst in accelerating a chemical reaction. The function, which depends on magnitude of the rate constants describing individual steps in the reaction, reaches a limiting value of unity when the reaction rate is controlled by diffusion. For the interconversion of dihydroxacetone phosphate and glyceraldehyde 3-phosphate, the efficiency function equals 2.5 x 10 for a simple carboxylate catalyst in a nonenzymic process and 0.6 for the enzyme-catalyzed process. Albery and Knowles suggest that evolution has produced a nearly perfect catalyst in the form of triose-phosphate isomerase. See Reaction Coordinate Diagram... 

In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate. 

FIGURE 20-10 Third stage of C02 assimilation. This schematic diagram shows the interconversions of triose phosphates and pentose phosphates. Black dots represent the number of carbons in each compound. The starting materials are glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Reactions catalyzed by transaldolase ( and ) and transketolase ((3) and ) produce pentose phosphates that are converted to ribulose 1,5-bisphosphate—ribose... 

One molecule of glyceraldehyde 3-phosphate is the net product of the carbon assimilation pathway. The other live triose phosphate molecules (15 carbons) are rearranged in steps to (S) of Figure 20-10 to form three molecules of ribulose 1,5-bisphosphate (15 carbons). The last step in this conversion requires one ATP per ribulose 1,5-bisphosphate, or a total of three ATP. Thus, in summary, for every molecule of triose phosphate produced by photosynthetic C02 assimilation, six NADPH and nine ATP are required. 

NADPH and ATP are produced in the light-dependent reactions of photosynthesis in about the same ratio (2 3) as they are consumed in the Calvin cycle. Nine ATP molecules are converted to ADP and phosphate in the generation of a molecule of triose phosphate eight of the phosphates are released as Pj and combined with eight ADP to regenerate ATP. The ninth phosphate is incorporated into the triose phosphate itself. To convert the ninth ADP to ATP, a molecule of Pj must be imported from the cytosol, as we shall see. 

FIGURE 20-14 Stoichiometry of C02 assimilation in the Calvin cycle. For every three C02 molecules fixed, one molecule of triose phosphate (glyceraldehyde 3-phosphate) is produced and nine ATP and six NADPH are consumed. 

Sucrose synthesis in the cytosol and starch synthesis in the chloroplast are the major pathways by which the excess triose phosphate from photosynthesis is harvested. Sucrose synthesis (described below) releases four Pi molecules from the four triose phosphates required to make sucrose. For every molecule of triose phosphate removed from the chloroplast, one Pj is transported into the chloroplast, providing the ninth Pj mentioned above, to be used in regenerating ATP. If this exchange were blocked, triose phosphate synthesis would quickly deplete the available Pj in the chloroplast, slowing ATP synthesis and suppressing assimilation of C02 into starch. 

Stromal enzymes, including transketolase and transaldolase, rearrange the carbon skeletons of triose phosphates, generating intermediates of three, four, five, six, and seven carbons and eventually yielding pentose phosphates. 

Conversion of Triose Phosphates to Sucrose and Starch Is Tightly Regulated... 

The flow of triose phosphates into sucrose is regulated by the activity of fructose 1,6-bisphosphatase (FBPase-1) and the enzyme that effectively reverses its action, PPrdependent phosphofructokinase (PP-PFK-1 p. 527). These enzymes are therefore critical points for determining the fate of triose phosphates produced by photosynthesis. Both enzymes are regulated by fructose 2,6-bisphosphate (F2,6BP), which inhibits FBPase-1 and stimulates PP-PFK-1. In vascular plants, the concentration of F2,6BP varies inversely with the rate of photosynthesis (Fig. 20-26). Phosphofructokinase-2,... 

The eight-stranded P cylinder of plastocyanin (Fig. 2-16A) is somewhat flattened and can also be regarded as a P sandwich.116118 However, the P barrel of triose phosphate isomerase (see Fig. 2-28) is surrounded by eight a helices which provide additional stability and a high symmetry. Bacterial outer membranes contain pores created by very large P cylinders within proteins called porins.119120 Tire one shown in Fig. 8-20 has 16 strands. 

Figure 2-28 The eight-fold oc/(3 barrel structure of triose phosphate isomerase. From Richardson. (A) Stereoscopic view. (B) Ribbon drawing. Courtesy of Jane Richardson.117...

Figure 13-6 Stereoscopic view into the active site of triose phosphate isomerase showing side chains of some charged residues PGH, a molecule of bound phosphoglycolohydroxamate, an analog of the substrate enolate.138 The peptide backbone, as an alpha-carbon plot, is shown in light lines.147 The (a/ (S)8-barrel structure is often called a TIM barrel because of its discovery in this enzyme. Courtesy of M. Karplus.

Another detail should be mentioned. The active site of triose phosphate isomerase is formed by a series of loops connecting the a helices and (3 strands of the barrel. One of those loops, consisting of residues 167-176, folds over the active site after the substrate is bound to form a hinged lid that helps to hold the substrate in the correct orientation for reaction.150-152 When the lid, which can be seen in Fig. 13-6, closes, the peptide NH of G171 forms a hydrogen bond to a phosphate oxygen atom of the substrate. This is only one of many known enzymes with deeply buried active sites that close in some similar fashion before a rapid reaction occurs. 

The rather toxic methylglyoxal is formed in many organisms and within human tissues.174 It arises in part as a side reaction of triose phosphate isomerase (Eq. 13-28) and also from oxidation of acetone (Eq. 17-7) or aminoacetone, a metabolite of threonine (Chapter 24).175 In addition, yeast and some bacteria, including E. coli, have a methylglyoxal synthase that converts dihydroxyacetone to methylglyoxal, apparently using a mechanism similar to that of triose phosphate isomerase. It presumably forms enediolate 2 of Eq. 13-26, which eliminates inorganic phosphate to yield methyl-... 

Formation of pyruvate. The conversion of glucose to pyruvate requires ten enzymes (Fig. 17-7), and the sequence can be divided into four stages preparation for chain cleavage (reactions 1-3), cleavage and equilibration of triose phosphates (reactions 4 and 5), oxidative generation of ATP (reactions 6 and 7), and conversion of 3-phosphoglycerate to pyruvate (reactions 8-10). 

The details of the process and the oxidation-reduction balance can be pictured as in Eq. 17-25. Pyruvate is cleaved by the pyruvate formate-lyase reaction (Eq. 15-37) to acetyl-CoA and formic acid. Half of the acetyl-CoA is cleaved to acetate via acetyl-P with generation of ATP, while the other half is reduced in two steps to ethanol using the two molecules of NADH produced in the initial oxidation of triose phosphate (Eq. 17-25). The overall energy yield is three molecules of ATP per glucose. The "efficiency" is thus (3 x 34.5)... 

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