Enzymes triose phosphate dehydrogenase

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. 

A detailed thermod5mamic analysis was performed with lactate dehydrogenase, in the lactate — p5mivate direction, by means of steady-state kinetics and presteady-state kinetic methods, by Laidler and Peterman (1979). A particularly detailed kinetic studies of the energetics of two multistep enzymes, triose-phosphate isomerase and prohne racemase, has been described by the research team of Albery and Knowles (Albery Knowles, 1976, 1986 Knowles, 1991). Apart from these examples, very few complete thermodynamic analyses have been performed with reactions involving more than one substrate or more than one intermediate in reaction. 

Another enzyme of the carbon reduction cycle activates PGA, converting it to phosphoryl-3-phosphoglyceric acid (IV). This acid anhydride can then be reduced in a subsequent enzymatic step mediated by triose phosphate dehydrogenase. For its reducing agent, this enzyme uses nicotinamide adenine dinucleotide phosphate (NADPH) and thereby converts the carboxylic acid to... 

Reactions (9), (10), and (13)-(15) bring about the conversion of PGA to fructose-6-phosphate (VI). The corresponding glycolytic enzymes would be (9) phosphoglyceryl kinase (10) triose phosphate dehydrogenase (13) triose phosphate isomerase (14) aldolase. Equation (15) would require a phosphatase. 

Triose phosphate dehydrogenase has been crystallized from rabbit muscle, and even after repeated crystallization, it contains two moles of bound NAD per mole of protein. The nucleotide can be removed by passing the enzyme through charcoal, but this treatment renders the enzyme preparation unstable. Both the stability and the activity of the enzyme can be restored by adding NAD to the medium. The presence of NAD in the enzyme molecule can readily be demonstrated by ultraviolet spectrophotometry. The exact mode of attachment of NAD to the enzyme molecule is not known, but it has been established that the enzyme contains SH groups belonging to cysteine residues—cysteine probably is part of the tripeptide gluta-... 

Triose phosphate dehydrogenase has been studied both as a chemical molecule and as a catalyst. These studies were made possible by the availability of gram quantities of crystalline enzyme from rabbit muscle, by the procedure of Cori, Slein, and Cori, and from yeast, by the procedure of Warburg and Christian. Krebs has isolated four fractions from yeast with equivalent specific activity, one of which is the enzyme crystallized by Warburg and Christian. The four components make up about 5 per cent of the total extractable protein of the yeast. 

Composition of Triose Phosphate Dehydrogenase. The animal and yeast enz3qnes are similar but not identical in certain respects. The amino acid compositions are similar, and both have N-terminal valine residues. The two are distinct immunologically. The most striking difference is the imusual binding of DPN in the rabbit enzyme. After repeated recrystallizations, each mole of this protdn contains two moles of firmly bound DPN. This DPN can be removed by treatment of the enzyme with charcoal. The charcoal-treated enzyme is less stable and more soluble than the original enzyme. 

Reactions of Triose Phosphate Dehydrogenase. This enzyme is not specific in its reaction with phosphoglyceraldehyde. The nonphospho-rylated compound is oxidized also, but only at 0.1 per cent of the rate found with the natural substrate. Acetaldehyde, propionaldehyde and butyraldehyde are also oxidized, but at still slower rates. The corresponding acyl phosphates are formed when these substrates are oxidized in the presence of inorganic phosphate. If arsenate is substituted for phosphate, the reactions proceed not to equilibrium, but to completion, with the formation of free acids. The formation of free acids is presumed to be the result of rapid spontaneous hydrolysis of unstable acyl arsenates, as proposed for other arsenolysis reactions. 

Triose Phosphate Dehydrogenases in Plants. Triose phosphate dehydrogenase, as described earlier, exists in many organisms. In green plants two additional enzymes have been reported. One is a very similar enzyme that has TPN in place of DPN. The other also uses TPN, but has no requirement for inorganic phosphate or any other acyl acceptor. This enzyme apparently catalyzes an irreversible formation of phospho-glyceric acid. 

As most of you know, when mammalian triose phosphate dehydrogenase (TPD) is crystallized, DPN is found to be firmly attached to the protein. The enzyme can be crystallized in either an active (SH) or inactive (S—S) form, both having the same low dissociation constant for the bound DPN. In addition, the active enz3rme may be obtained with bound DPNH. 

Heavy metals frequently act as enzyme inhibitors particularly when free -SH groups participate in the reaction. Papain, urease, myosin, triose phosphate dehydrogenase and many other enzymes fall into this category and are readily inactivated by Ca, Hg " " etc. The inhibition may sometimes be overcome by removing the metal ion, e.g. with H2S or with a chelating agent such as EDTA. 

Fig. 36. The Calvin cycle (black lines) and pentose phosphate cycle (red lines). PGA = 3-Phosphoglyceric acid, PGAL = 3-phosphoglyceraldehyde, Rib. = ribose-5-phosphate, Xyl = xylulose-5-phosphate, Ru-diP = ribulose-1,5-diphosphate, C4 = erythrose-4-phosphate, FDP = fructose-1,6-diphosphate. A few of the enzymes participating are encoded, 1 = carboxydismutase, 2 = triose phosphate dehydrogenase, 3 = triose phosphate isomerase, 4 = aldolase, 5 = phosphatase, 6 = phosphoglucoisomerase. Details of the conversion of glucose-6-P into ribulose-5-P are given in Fig. 43. It should be pointed out that the pentose phosphate cycle presents only here and there a true reversal of the Calvin cycle. In many instances the mechanisms and enzymes are different.

Since triose phosphate dehydrogenase is a key enzyme in carbohydrate metabolism, particular importance was attached to its identification in leaves. The experiments of Bonner and Wildman on minced spinach leaves suggested the presence of this enzyme. 

Of possible significance for their role in photosjmthetic tissues is the fact that the three reductive carboxylases described above are all TPN-specific. Even though similar DPN-linked enzymes are known, a net fixation of CO has as yet been observed only in the TPN-linked reactions. This dependence of CO fixation on TPN, like the occurrence of a TPN-linked triose phosphate dehydrogenase in photosynthetic tissues, may be related to the TPN specificity of chloroplasts (190). 

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