Direct Competitive Kinetic Isotope Effect Measurement Using Quantitative Whole Molecule Matrix-Assisted Laser...

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Kinetic isotope effect (KIE) measurements are crucial for understanding enzyme mechanisms and transition states.
• Current KIE measurement methods are often complex and time-consuming, limiting their widespread application.
• Direct measurement of competitive KIEs in enzymatic reactions requires robust analytical techniques.

Purpose of the Study:

• To develop a simplified and direct method for measuring competitive kinetic isotope effects (KIEs) in enzyme-catalyzed reactions.
• To apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for precise KIE determination.
• To investigate the hydrolysis of lactose by E. coli β-galactosidase (LacZ) using the developed method.

Main Methods:

• Developed a novel method utilizing whole molecule MALDI-TOF MS for direct KIE measurement.
• Incorporated an isotope-labeled internal standard added at reaction quench points.
• Simultaneously measured relative isotope ratios (R) and fractional conversion (F) over time.
• Applied the method to determine KIEs for [1'-¹³C]lactose and [6'-¹³C]lactose in E. coli β-galactosidase catalyzed hydrolysis.

Main Results:

• Successfully implemented a direct MALDI-TOF MS approach for competitive KIE measurements.
• Achieved precise KIE measurements comparable to established radioisotope labeling and NMR methods.
• Quantified KIEs for specific carbon isotopes ([1'-¹³C] and [6'-¹³C]) in lactose hydrolysis.
• Demonstrated the method's utility for studying E. coli β-galactosidase kinetics.

Conclusions:

• The developed MALDI-TOF MS method offers a direct, precise, and efficient way to measure enzymatic KIEs.
• This technique simplifies the analytical workflow, making KIE studies more accessible.
• The approach provides valuable insights into the microscopic steps of enzyme-catalyzed reactions, aiding in transition state analysis.