Page 341 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

15.2 Cascade Reactions for Assaying Transketolase Activity In Vitro 317

Colorimetric assay Colorimetric assay
R′ 1 = CO 2 R′ 1 = CO 2
R 3 = H,
R = Aliphatic or aromatic
4
Spectrophotometric

Phenol red assay
Formazane ADH
TPI R′ 1 = CHO-CHOH-R 2
R 2 = CH 2 OP
O R 3 O R 3
TK
HO + HO + BSA Fluorimetric assay

R R 2+ R 4 R′ 1 R′ = CHO-CHOH-R
1 4 ThDP, Mg 1 2
O OH R = Umbelliferone
2
BSA
R 1 = CO 2 H, CHOH-CHOH-R 2 PPO
E. coli
auxotrophs Amperometric assay
R′ 1 = CHO-CHOH-R 2
In vivo assay R = Protected tyrosine
2
1
R′ = CHO-CHOH-R 2
R 2 = CH 2 CH(CH 3 ) 2 , (CH 2 ) 2 SCH 3
Scheme 15.3 TK assays based on cascade reactions. ADH, alcohol dehydrogenase; TPI,
triose phosphate isomerase; BSA, bovine serum albumin; PPO, polyphenoloxidase.
15.2
Cascade Reactions for Assaying Transketolase Activity In Vitro
There have been several in vitro assay methods reported recently that are based
on spectrophotometric, fluorometric, or amperometric means of detection. These
assays were investigated using various natural, commercially available substrates
or from specially designed and synthesized probes. The prerequisite for the donor
substrate is a ketol moiety, and for the acceptor substrate an aldehyde function.
Substrates or products of the TK reaction cannot be specifically quantified by
any molecular property, which requires a subsequent specific chemical or enzyme-
catalyzed cascade reaction to create a measurable signal for specific quantification.
Such coupled assays can be classified according to the nature of the auxiliary
agent involved in the cascade reaction. Depending on the principle of the assay,
continuous measurements or only discontinuous end-point determinations can
be made.

15.2.1
Coupling with Other Enzymes as Auxiliary Agents

15.2.1.1 Coupling with NAD(H)-Dependent Dehydrogenases
The conventional method for measuring TK activity uses d-ribose 5-phosphate
(d-R5P) as acceptor and d-xylulose 5-phosphate (d-X5P) as a donor [15] because
these compounds are high affinity natural substrates of TK that support highest
reaction rates. d-Glyceraldehyde 3-phosphate (d-G3P) generated upon cleavage
of the two-carbon unit from this donor substrate can subsequently be intercon-
verted with dihydroxyacetone phosphate (DHAP) using triose phosphate isomerase
(TPI) (Scheme 15.4), and then reduced into d-glycerol 3-phosphate using NADH