Triosephosphate isomerase: a highly evolved biocatalyst.

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Triosephosphate isomerase (TIM) is a highly efficient enzyme catalyzing the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate.
• The active site of TIM is located at the dimer interface, with key catalytic residues including Asn11, Lys13, His95, and Glu167, all originating from the same subunit.
• Glu167 serves as the catalytic base in the TIM reaction mechanism.

Purpose of the Study:

• To elucidate the role of loop-6 and loop-7 closure in shielding the TIM active site.
• To investigate the dynamic properties of the Glu167 side chain in the enzyme-substrate complex.
• To understand the proton shuttling mechanisms responsible for substrate interconversion.

Main Methods:

• Analysis of enzyme structure and active site dynamics.
• Focus on the movement of catalytic residue Glu167 upon ligand binding.
• Examination of loop-6 and loop-7 conformational changes.

Main Results:

• Ligand binding induces concerted closure of loop-6 and loop-7, shielding the active site and stabilizing the enediolate intermediate.
• The catalytic residue Glu167 moves approximately 2 Å upon loop-6 closure, positioning it optimally for catalysis.
• Dynamic properties of the Glu167 side chain are crucial for the proton shuttling mechanism.

Conclusions:

• The active site shielding mechanism involving loop closures is critical for TIM's catalytic efficiency.
• Glu167's dynamic repositioning is a key feature of the proton shuttling process.
• Both classical and criss-cross mechanisms contribute to the substrate interconversion catalyzed by TIM.