Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O

摘要

Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonylncarbon ([1-n13nC]-GA) catalyzed by triosephosphate isomerase (TIM) in D2O at pD 7.0 in the presence ofnphosphite dianion and in its absence were determined by 1nH NMR spectroscopy.We observe three productsnfor the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46,n5841-5854): [2-n13nC]-GA from isomerization with intramolecular transfer of hydrogen (12% of products),n[2-n13nC,2-n2nH]-GA from isomerization with incorporation of deuterium from D2O at C-2 (64% of products),nand [1-n13nC,2-n2nH]-GA from incorporation of deuteriumfrom D2O at C-2 (23%of products). Themuch slowernunactivated reaction in the absence of phosphite results in formation of the same three products along with thendoubly deuterated product [1-n13nC,2,2-n2nH2]-GA. The two isomerization products ([2-n13nC]-GA andn[2-n13nC,2-n2nH]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activatednreactions.However, the additional [1-n13nC,2-n2nH]-GA and the doubly deuterated [1-n13nC,2,2-n2nH2]-GA formed innthe unactivated TIM-catalyzed reaction are proposed to result from nonspecific reaction(s) at the proteinnsurface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution,nof the TIM-catalyzed reaction of [1-n13nC]-GA at the enzyme active site. They are consistent with the conclusionnthat both reactions occur at an unstable loop-closed form of TIM and that activation of the isomerizationnreaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianionnto stabilize the active loop-closed enzyme.