Watching Alkaline Phosphatase Catalysis Through Its Vibrational Fingerprint.

Area of Science:

• Biochemistry
• Spectroscopy
• Enzymology

Background:

• Alkaline phosphatase (ALP) catalysis mechanisms are well-studied structurally and kinetically.
• However, the molecular vibrations during ALP catalysis remain largely unexplored.

Purpose of the Study:

• To explore the spectral molecular vibrations during alkaline phosphatase (ALP) catalyzed hydrolysis.
• To develop a generalizable approach for tracking catalytic processes using FTIR.

Main Methods:

• In situ real-time attenuated total reflection Fourier transform infrared (ATR-FTIR) measurements were performed.
• Hydrolysis of p-nitrophenyl phosphate (PNPP) by ALP was monitored over a range of enzyme concentrations.
• Static spectra of pure components (ALP, PNPP, PNP, Pi) were used as references.

Main Results:

• The inorganic phosphate (Pi) band at 1077 cm⁻¹ showed monotonic growth.
• Specific blue and red shifts in vibrational frequencies were observed in the nitro/aromatic and fingerprint regions, respectively.
• A splitting of the ~1592 cm⁻¹ band was resolved at high enzyme concentrations.

Conclusions:

• The study successfully resolved the infrared readout of the enzymatic reaction by anchoring time-resolved spectra to static spectra.
• This provides a generalizable FTIR-based approach for tracking catalytic processes.