A general method for the assay of thiamine pyrophosphokinase activity

Thiamine diphosphate (ThDP) is a coenzyme for several essential enzymes in cell metabo-lism (mainly transketolase, pyruvate and 2-oxoglutarate dehydrogenases). In eukaryotic cells ThDP is synthesized by pyrophosphorylation of thiamine (vitamin B1) by ATP or UTP and cata-lyzed by thiamine pyrophosphokinase isoform 1 (TPK1). Complete inactivation of TPK1 is lethal, but recently several hereditary mutations in the corresponding gene were described in human families. The phenotype is generally severe with acute encephalopathy and fatal outcome when not treated with excess thiamine. This suggests that the mutated enzyme still retains a fraction of its native activity. In order to estimate the effect of the mutations on enzyme activity it is essential to benefit from a reliable assay of TPK1 activity. This enzyme has a low KM for thiamine (KM < 0.1 µM) but a low turnover (kcat = 0.03 s-1) requiring very sensitive methods for the detection of the product ThDP by HPLC followed by fluorometric (after derivatization into fluorescent thio-chrome derivatives) or mass spectrometric detection. Moreover, ThDP is a noncompetitive inhibi-tor of TPK1 with a KI = 0.4 µM. Hence, when measuring TPK1 activity care must be taken that the reaction is stopped before inhibition becomes significant. Here, we present a powerful HPLC method coupled to fluorometric detection allowing TPK1 assay either in the forward or the re-verse direction. The importance of such a method is further highlighted by recent data showing decreased expression of TPK1 in blood and brain of Alzheimer patients making TPK1 assay an interesting alternative to the estimation of TPK1 expression.