Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Triosephosphate isomerase (TIM) catalyzes DHAP/GAP interconversion.
• Active-site residue Glu165 acts as the catalytic base.
• Phosphate binding influences TIM's catalytic mechanism.

Purpose of the Study:

• To investigate the role of phosphate binding in TIM catalysis.
• To determine the pKa elevation of the catalytic base Glu165.
• To elucidate the deprotonation kinetics of Glu165.

Main Methods:

• Difference Fourier transform infrared spectroscopy (FTIR) on yeast TIM (YeTIM)/phosphate complex.
• Infrared (IR) T-jump studies on YeTIM/phosphate and heavy enzyme complexes.
• Kinetic isotope effect (KIE) analysis.

Main Results:

• Phosphate binding increases Glu165 pKa by >3.0 pH units.
• Phosphate binding maintains a hydrophilic environment around Glu165.
• Two deprotonation kinetics (μs and ms) observed for Glu165.
• Normal KIE (1.2) for ms deprotonation (phosphate dissociation).
• Inverse KIE (0.89) for μs deprotonation (pKa decrease).

Conclusions:

• Phosphate binding is integral to TIM's activation mechanism via pKa elevation.
• Enzyme mass and loop motion influence phosphate dissociation kinetics.
• IR spectroscopy provides atomic-level insights into TIM's catalytic mechanism.