Role of hydrophobic loop residues in catalysis by triosephosphate isomerase
Malabanan, Maria Merced U.
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The role of two selected hydrophobic residues, Isoleucine-172 and Leucine-232, in catalysis by triosephosphate isomerase was investigated. Mutations of these residues into a smaller methyl group and subjecting the resulting mutants to the following mechanistic probes provide valuable information on the mechanism of the TIM-catalyzed reaction: (1) Experiments to determine the yields of the three products from the TIM-catalyzed reaction of glyceraldehyde 3-phosphate (GAP) in D2O; (2) Experiments to determine the kinetic parameters for activation of TIM-catalyzed deprotonation of [1- 13 C]-glycolaldehyde ([1- 13 C]-GA) by phosphite dianion; (3) Experiments to determine the yields of the products of the unactivated and phosphite-activated reactions of [1- 13 C]-GA in D 2 O; and (4) Experiments to determine the variation on pH of the kinetic parameters for the isomerization of the substrate GAP and on inhibition of wildtype and I172A mutant TIM by phosphoglycolate. We observed that the L232A mutation resulted in an increase in the efficiency of TIM in catalyzing proton transfer reaction of the substrate pieces [1- 13 C]-GA and phosphite, indicating that this residue plays an important role in the activation of TIM for deprotonation at carbon. The I172A mutation resulted in a decrease in the basicity of the catalytic base Glu-167, indicating that this residue plays an important role in the reactivity of TIM towards isomerization reaction.