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Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural
W Schliebs1, N Thanki, R Eritja
1European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.
Insights
MonoTIM, a monomeric variant of trypanosomal triose phosphate isomerase (TIM), retains catalytic activity despite altered flexibility. Key residues Lys 13 and His 95 are essential for monoTIM
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Trypanosomal triose phosphate isomerase (TIM) is a dimeric enzyme crucial for glycolysis.
- MonoTIM is a stable monomeric variant with reduced but significant catalytic activity.
- Key catalytic residues Lys 13, His 95, and Glu 167 are known in wild-type TIM.
Purpose of the Study:
- To investigate the role of Lys 13 and His 95 in monoTIM catalysis and substrate binding.
- To elucidate the structural basis of monoTIM's catalytic mechanism using substrate analogues.
Main Methods:
- Site-directed mutagenesis to create monoTIM variants (K13A, H95A).
- Enzyme kinetics to assess catalytic activity and inhibitor binding affinity (KI values).
- Protein crystallography to determine the binding mode of phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG).
Main Results:
- MonoTIM-K13A mutant is inactive but retains substrate analogue binding.
- MonoTIM-H95A mutant shows a 50-fold decrease in activity.
- MonoTIM exhibits reduced affinity for PGH (60-fold) but similar affinity for 2PG (2-fold) compared to wild-type TIM.
- Crystallographic analysis reveals 2PG interacts with Lys 13 and His 95, and Glu 167 maintains interactions similar to wild-type TIM.
Conclusions:
- Lys 13 and His 95 are essential for optimal monoTIM catalysis, despite increased flexibility.
- MonoTIM utilizes a catalytic mechanism similar to wild-type TIM, even without the second subunit.
- Structural insights confirm the conserved role of Glu 167 and highlight specific interactions of 2PG with active site residues.