Enzymic methods transketolase

In a very imaginative piece of research Frost and coworkers have developed a plasmid-based method for synthesizing aromatic amino acids, by incorporating the genes that code for the enzymes that perform the series of conversions from D-fructose-6-phosphate to D-erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonic acid-7-phos-phate (DAHP) near each other on a plasmid that can be transformed in E. coli. The enzymes are the thiamin diphosphate-dependent enzyme transketolase in the nonoxida-tive pentose shunt and DAHP synthase. The DAHP is then converted to the cyclic dehydroquinate, a precursor to all aromatic amino acids L-Tyr, L-Phe and L-Trp165,166 (equation 27). 

The most reliable method for assessing thiamin status involves the measurement of red blood cell transketolase. This enzyme is measured with and without the addition of TPP to the enzyme assay mixtures. In dietary thiamin deficiency, synthesis of transketolasc continues, but conversion of the apoet zyme to the holoenzyme in the cell is inhibited, resulting in the accumulation of the enzyme in the apoenzyme form. Addition of TPP to cell homogenates results in the conversion of apoenzyme to holoenzyme. This conversion can easily be detected by enzyme assays. The amount of shmulation of enzyme activity by the added TPP is used to assess thiamin status. A deficiency is indicated by a shmulation of over 20%, The TPP-dependent stimulation, using red blood cells from normal subjects, ranges from 0 to 15%. 

Standard methods for assessment of thiamine status used to be determination of erythrocyte transketolase (a-ETK) activity (EC 2.2.1.1) with and without stimulation of this enzyme by addition of TDP cofactor (TOP TK effect). A TDP TK effect >15% is considered to show some degree of deficiency, whereas values >22% are considered to indicate severe deficiency. Technical difficulties, including standardization of the assay, instability of the enzyme during storage, and various conditions possibly influencing apoenzyme concentrations led to an increasing use of direct determination of TDP in whole blood, e.g., by HPLC in order to assess thiamine status. The HPLC assay is more robust and easier to perform. Thiamine... 

ETK based methods, once considered the most reliable means of assessing thiamine status, are now considered inadequate because they only provide an indirect measure. Because transketolase activity requires thiamine, decreased transketolase activity is presumed to be due to a decrease in thiamine. However, other factors may decrease transketolase activity including decreased enzymatic binding and decreased enzyme synthesis as has been demonstrated in diseases such as diabetes (Friedrich 1988) and liver dysfunction (Feimelly et al. 1967). ETK based methods have also been criticized as unreliable, insensitive, and subject to poor precision (Bailey et al. 1994). 

A rapid, high yield synthesis of C-enriched intermediates of the pentose-phosphate pathway has been developed based on a combination of chemical and enzymic reactions. [l- C]Ribose and [l- C]arbinose 5-phospha,tes, available by the classical Kiliani method, were converted to a variety of specifically labelled 5-, 6-, 7-, and 8-carbon sugar phosphates (e.g.. D-erythro-pentulosc 5-phosphate, sedoheptulose mono- and di-phosphates) with the help of aldolase, transaldolase, and transketolase. ... 

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