Determination method for activity of enzymes like farnesyl pyrophosphate synthase

Abstract

So far, activity of enzymes can only be detected by using radio isotope or fluorophore labeling methods when substrates of the enzymes do not have ultraviolet-visible absorption. The detection methods have the disadvantages of high cost, unfriendliness to the environment and difficult operation. The invention provides a determination method for the activity of enzymes like farnesyl pyrophosphate synthase. The method is characterized by using colourimetry to detect a produced product--pyrophosphoric acid or phosphoric acid, then reacting pyrophosphoric acid or phosphoric acid with a specific reagent and carrying out reduction so as to obtain a colored compound. The determination method has the advantages of high efficiency, sensitivity, economic performance, etc. The determination method has broad spectrum activity, can be used for detecting the activity of geranylgeranyl pyrophosphate synthetase (GGPPS), squalene synthetase (SS), farnesyl transferase (FTase), geranylgeranyl transferase (GGTase), mevalonate pyrophosphate decarboxylase (MDD), etc. except the activity of farnesyl pyrophosphate synthase, and is applicable to high flux screening of enzyme inhibitors and development of drugs.

Description

technical field [0001] The invention relates to a method for measuring the activity of enzymes such as farnesyl pyrophosphate synthase, and belongs to the technical field of enzymatic detection. Background technique [0002] Phosphoric acid or pyrophosphate plays an important role in the metabolism of organisms, and many enzymes in organisms use or generate phosphoric acid or pyrophosphate in catalytic reactions. For example, farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase (GGPPS), squalene synthase (SS), farnesyltransferase (FTase), geranylgeranyltransferase (GGTase), mevalonate pyrophosphate decarboxylase (MDD), etc. These enzymes are important enzymes in the Mevalonate pathway. Mevalonate pathway (Mevalonate pathway) is a metabolic pathway for the synthesis of isoprene pyrophosphate and dimethylallyl pyrophosphate from acetyl-CoA, which exists in all higher eukaryotes and many viruses. The products of this pathway can be viewed as activated isopr...

AI Technical Summary

Problems solved by technology

The former requires expensive radioactive substrates to be detected, and cannot be operated continuously, the steps are complicated, and it is also more harmful to the environment

The latter method requires unnecessary and precise modification of the fluorescent group on the substrate, and it is possible to detect it, and it has high requirements for the detection system, many interference factors, and low accuracy. Some systems are not suitable for aqueous solution operation.

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