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  • Hydron transfer catalyzed by triosephosphate isomerase. Products of the direct and phosphite-activated isomerization of [1-(13)C]-glycolaldehyde in D(2)O.

Hydron transfer catalyzed by triosephosphate isomerase. Products of the direct and phosphite-activated isomerization of [1-(13)C]-glycolaldehyde in D(2)O.

Biochemistry (2009-05-12)
Maybelle K Go, Tina L Amyes, John P Richard
ABSTRACT

Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) catalyzed by triosephosphate isomerase (TIM) in D(2)O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-(13)C]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-(13)C,2-(2)H]-GA from isomerization with incorporation of deuterium from D(2)O at C-2 (64% of products), and [1-(13)C,2-(2)H]-GA from incorporation of deuterium from D(2)O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-(13)C,2,2-(2)H(2)]-GA. The two isomerization products ([2-(13)C]-GA and [2-(13)C,2-(2)H]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions. However, the additional [1-(13)C,2-(2)H]-GA and the doubly deuterated [1-(13)C,2,2-(2)H(2)]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-(13)C]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.

MATERIALS
Product Number
Brand
Product Description

Supelco
AmberChrom 50WX4 hydrogen form, hydrogen form, 50-100 mesh
Supelco
AmberChrom 50WX4 hydrogen form, hydrogen form, 100-200 mesh
Supelco
AmberChrom 50WX4 hydrogen form, 200-400 mesh, Cation Exchange Resin