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HO- O : H O - OH TPP

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Figure 1.6. Proposed reaction mechanism for transketolase catalysis (Kluger, 1990).

The TPP is bound to the N-terminal domain of transketolase and interacts with the protein through hydrogen bonding and indirectly via the divalent metal cation. The metal ion acts as an additional anchor for the TPP. Leading to the bound cofactor is a channel or cleft, formed by loop regions of the proteins, which binds and orientates the substrates (Schneider and Lindqvist, 1993). No large conformational changes are associated with cofactor binding but it is postulated that there are two loops at the entrance to the active site cleft which are flexible enough to allow TPP and the metal cation to reach the active site. Once the cofactors are bound, the two loops interact with each other and the cofactors stabilise the conformation (Sundstrom et al., 1992). Several divalent metal cations may act as a cofactor for yeast transketolase (Kotchetov and Meshalkina, 1979). In order of decreasing activity:

» Z n * /

The presence of a phosphate binding site for TPP interaction may explain the observation that both sulphate and phosphate inhibit the trasketolase reaction at >5 mM concentration (Datta and Racker, 1961; Kotchetov, 1982).

A potentially unlimited source o f transketolase has recently been reported by several workers using recombinant E. coli (Sprenger, 1991, Hobbs et a l, 1993). In both these cases the chromosomal transketolase gene from E coli was transformed back into E. coli at multiple copy number. Yield increases of thirty to forty fold over parent have been reported (Sprenger et al., 1995). The crystalline structure of this E. coli transketolase has been elucidated by Littlechild and co-workers (1995). and found to show 43% sequence homology to the yeast enzyme. As the active site residues appears to be almost identical to the yeast enzyme, the reaction mechanism for the yeast and E. coli derived enzymes is probably also similar.

1.4.4. Measurement of transketolase activity

Several assays for transketolase have been reported in the literature. The most widely used assays use the natural phosphorylated sugar substrates and link the formation of product to a further dehydrogenase catalysed reaction. The transketolase is diluted such that this enzyme is rate limiting in the cascade reaction and the loss of NADH in the dehydrogenase step is monitored spectrophotometrically (Heinrick et al., 1972; Villafranca et al., 1971; Hecquet et al., 1993). The main disadvantage with these assays are the high cost of both the substrates and additional enzymes.

The use of hydroxypyruvate (HPA) as the ketol donor in the transketolase reaction provides several theoretical routes for measurement of activity. The loss of HP A can be monitored directly by absorbance at 210-240 nm (Kotchetov and Phillipov, 1972) or enzymatically using glycerate dehydrogenase and alcohol dehydrogenase linked to NADH oxidation (Holldorf, 1966). The direct spetrophotometric method is not suitable for low levels of activity due to the high initial absorbance. Enzymatic determination of HP A concentration requires the transketolase to be denatured by acidification and the pH re-adjusted back to near neutral. This introduces significant errors into the system particularly with samples of low volume. Alternatively, the utilisation of HP A results in an increase in pH which can be monitored spectrophotometrically using colorimetric indicators such as p-nitrophenol (Hubner et al., 1992). However, in practice, these absorbance changes are too small to be measured accurately (Hobbs, 1994). A coupled enzyme system using phospho- enol-pyruvate carboxylase (PEPC) linked to malate dehydrogenase (MDH) has also been reported (Burns and Aberhart, 1988). Carbon dioxide evolved during the transketolase reaction is one of the substrates for PEPC, the product of which is a substrate for MDH resulting in reduction of NAD to NADH. However, the difficulty with this system is maintaining the various substrate and enzyme solutions completely free of extraneous dissolved carbon dioxide. Transketolase may also be assayed by monitoring hexacyanoferrate reduction in the presence o f a donor substrate (Usmanov and Kotchetov, 1991) although this is not a practical method as

without disadvantages. However, for research purposes, the measurement of transketolase using natural substrates via a multiple enzyme cascade appears to be the most accurate and the most suitable (Hobbs, 1994).

1.4.5. Choice of model transketolase reaction and process implications

In order to study the process leading to bioreactor design and operation for transketolase catalysed biotransformations, it is necessary to choose a model reaction. Commercial exploitation of transketolase for synthetic purposes is rarely concerned with reactions reaching equilibrium. The use of HP A as ketol donor results in irreversible reaction and stoichiometric production of carbon dioxide. Carbon dioxide production during reaction poses a number of potential process design problems. Gas production may result in foaming and denaturing of catalyst in addition to creating channelling and buoyancy effects in immobilised enzyme systems. However, reaction conditions can be manipulated to reduce these effects. The stoichiometric production of carbon dioxide during production of the amino acid L-alanine was reportedly eliminated by pressurising the reaction system (Furui and Yamashita, 1983; Senuma et al., 1989). These workers used immobilised Pseudomonas cells in a packed-bed reactor pressurised to 8 kg/cm^ . Chemical reactions with carbon dioxide may also be a potential problem. The dissociation products of carbon dioxide dissolution may react with proteins to produce carbamates (Figure 1.7) (Mitz, 1979; Fox, 1991) and may also result in protein/bicarbonate colloid formation (Paxton, 1974). Also, as mentioned previously, the use of an HP A salt as ketol donor results in a significant pH shift to alkali during reaction. Although carbon dioxide dissociation partially offsets this shift, measures to control the change in pH, particularly on a large scale, need to be considered.

R C COOH + CO-