A new method of determination of transketolase activity by asymmetric synthesis reaction

✍ Scribed by G.A. Kochetov; R.A. Usmanov; A.T. Mevkh

• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 298 KB
• Volume: 88
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90422-0

✦ Synopsis

A new method is proposed for assaying transketolase activity. The method is based on determining erythrulose, the product of the transketolase reaction, by its optical activity.

Transketolase (EC 2.2.1. l), an enzyme of the pentose pathway of hydrocarbon conversion, catalyzes the transfer of the ketol group from the donor substrate (ketose phosphate) to a suitable aldehyde acceptor (aldose phosphate). TK' may use as substrates simpler compounds containing no phosphate. group, i.e., hydroxypyruvate, L-erythrulose, and DL-xylulose as donors and glycolaldehyde and glyceraldehyde (1) as acceptors. The activity of TK may be measured by optical methods or determined chemically. The chemical method (2) enables one to directly determine the absolute quantity of the product, sedoheptulose 7-phosphate, formed in the transketolase reaction:

Xy-5-P + R-5-P = PGA + S-7-P.

The other method, optical, also allows one to dispense with the auxiliary enzymes (3) and follow the rate of transketolase reaction the decrease in optical density at 210 to 240 nm due to the disappearance of hydroxypyruvate in the reaction HP + R-5-P --, S-7-P + CO,.

The third method, the most handy in routine experiments, requires auxiliary enzymes. It permits one to follow the rate of the transketolase reaction by the optical density alteration at 340.nm (at the expense of NAD reduction or NADH oxidation). Nevertheless, this method proves insufficient in studies on the effect of temperature, pH, or various effecters that may also change the activity of auxiliary enzymes.