Triosephosphate isomerase isotope labeled

The substrates of triosephosphate isomerase can be labeled specifically with deuterium. The rate of the isomerase reaction with [l(R)-2H]-dihydroxyacetone phosphate (a) as substrate is slowed by about a factor of 3 relative to the rate with ordinary dihydroxyacetone phosphate. This isotope effect indicates that breaking the H—C bond to form the ene-diolate intermediate is the rate-limiting step in conversion of dihydroxyacetone phosphate to... 

Scheme 5.42. Synthesis of isotopically labeled nucleosides using a combined enzyme system. Conditions (a) TFAA/Br2 (b) BzlOH/NaH (c) (i) SOCl2) (a) CH2N2 (d) (Bzl0)2P(0)0H (e) H2 Pd/C. TPI = triosephosphate isomerase.

Figure 17-29. Synthesis of [3, 4 -13C2]-thymidine from [2, 3 -13C2]-dihydroxyacetone phosphate with triosephosphate isomerase (TPI) and D-2-deoxyribose-5-phosphate (DHAP). Asterisks indicate the positions selectively labeled with 13C. Other positions that can be isotopically substituted are marked with , A, and V. Reprinted from Ouwerkerk et al.12511.

Characterization of the product of the reaction of chloroacetol phosphate with triosephosphate isomerase was facilitated by reduction of the derivatized protein with sodium borotritride. Initially the only isotopically labeled chloroacetol phosphate available was the P-labeled material. 

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