A new method for continuous recording of the transketolase reaction

✍ Scribed by G.A. Kochetov; P.P. Philippov

• Publisher: Elsevier Science
• Year: 1972
• Tongue: English
• Weight: 289 KB
• Volume: 48
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(72)90191-1

✦ Synopsis

Transketolase (EC 2.2.1.1) is the enzyme that catalyzes the transfer of the two-carbon fragment, the so-called "active glycolaldehyde," from the donor substrate (xylulose &phosphate, fructose 6-phosphate, sedoheptulose 7-phosphate, hydroxypyruvate, etc.) to the acceptor substrate (ribose 5-phosphate, glyceraldehyde S-phosphate, erythrose 4-phosphate, etc.)

Transketolase activity is usually measured by means of one of three methods (1) :

According to one, samples should be chosen from the incubation mixture to determine the quantity of sedoheptulose 7-phosphate formed. In this way no continuous follow up of the enzymic reaction can be provided. Besides, determination of sedoheptulose 7-phosphate is a timeconsuming procedure and the results of the experiments cannot be readily appreciated.

According to the two other methods, auxiliary enzymes (glyceraldehydephosphate dehydrogenase or triosephosphate isomerase and glycerophosphate dehydrogenase) and necessary substrates and cofactors are added to the reaction mixture besides the components of the transketolase reaction itself (transketolase, a bivalent cation, thiamine pyrophosphate, xylulose 5-phosphate, and an acceptor substrate). These two methods make it possible to follow continuously the transketolase reaction measuring change in optical density at 340 nm (at the expense of either NAD reduction or oxidation of reduced NAD). These two methods do not allow investigation of the effect of some inhibitors (for example, ions of heavy metals) and reaction conditions (temperature, pH, etc.) upon the transketolase activity, as their effects may be due to the suppression of activity of the auxiliary enzymes.

In the present paper, a method is described allowing continuous measurement of the transketolase reaction without requiring auxiliary 286