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316 15 New Applications of Transketolase: Cascade Reactions for Assay Development

O OH OH
2−
HO OPO 3 R
+
OH O OH
2−
D-Xylulose-5-P R = -CH 2 OPO 3 : D-erythrose-4-P
2−
R = -CHOH(R)-CH 2 OPO 3 : D-ribose-5-P
ThDP, Mg 2+ TK

OH O OH
2−
OPO 3 + HO R
O OH OH
2−
D-Glyceraldehyde-3-P R = -CH 2 OPO 3 : D-fructose-6-P
2−
R = -CHOH(R)-CH 2 OPO 3 : D-sedoheptulose-7-P
Scheme 15.1 In vivo TK reaction.
O OH O OH
TK
HO + HO + CO 2
CO 2 H R ThDP, Mg 2+ R
O OH
HPA Ketose (3S,4R)

Scheme 15.2 Principle of synthetic applications of TK based on the decarboxylation of
hydroxypyruvate.

enzymatic activity against a structural variety of substrates to estimate their suit-
ability for preparative goals. The mapping of an enzyme’s substrate tolerance [8, 9],
also called activity fingerprinting [10, 11], is a valuable technique for characterizing
the scope of new synthetic applications of enzymes. In addition, the screening
of large libraries of mutant enzymes requires efficient in vitro high-throughput
screening (HTS) assays or the direct detection of enzyme activity in host cells using
in vivo assays, to obviate the need for cell lysis and purification of enzymes.
Some recent studies have shown that human TK is a target in various dis-
eases, suggesting new therapeutic approaches. Among the human proteome, TK
is reported to play a pivotal role in carcinogenesis, and may have important
implications for the nutrition and future treatment of patients suffering from
neurodegenerative diseases [12], diabetes [13], and cancer [14]. Therefore, the devel-
opment of rapid, sensitive, and efficient in vitro methods for TK activity profiling is
also highly desirable to enable the identification of suitable inhibitors.
In this context, new assays for TK activity detection have been recently devel-
oped. This chapter describes the recent in vivo and in vitro TK assays based on
cascade reactions involving one or more auxiliary enzymes and/or protein and/or
nonprotein agents (Scheme 15.3).