# Watching Alkaline Phosphatase Catalysis Through Its Vibrational Fingerprint

## TL;DR

The paper uses infrared spectroscopy to observe real-time molecular vibrations during alkaline phosphatase catalysis, revealing new insights into enzyme activity.

## Contribution

The study introduces a novel approach using time-resolved ATR-FTIR to track enzymatic reactions under near-native conditions.

## Key findings

- The reaction shows a monotonic growth of the inorganic-phosphate band at 1077 cm−1.
- Blue and red shifts in vibrational frequencies were observed in the nitro/aromatic and fingerprint regions.
- The ~1592 cm−1 band splits into two distinct peaks at 1595 and 1583 cm−1 at high enzyme concentrations.

## Abstract

Alkaline phosphatase is an important enzyme involved in many biological processes and is widely studied as a model system for enzyme catalysis. While its structure and activity have been extensively characterized, much less is known about how molecular vibrations evolve during the catalytic reaction. In this work, we use infrared spectroscopy to monitor in real time the conversion of p-nitrophenyl phosphate to p-nitrophenol by alkaline phosphatase in aqueous solution. By comparing time-resolved spectra with reference spectra of the enzyme, substrate, and products, we directly follow product formation and changes in vibrational features during catalysis. This approach provides a direct spectroscopic view of enzymatic activity and illustrates how infrared spectroscopy can be used to study catalytic processes under near-native conditions.

Despite decades of structural and kinetic characterization, the full spectral molecular vibrations that accompany the catalysis in alkaline phosphatase (ALP) have remained largely unexplored. In this study, we combine in situ real-time attenuated total reflection Fourier transform infrared (ATR-FTIR) measurements over a large energy range to track the hydrolysis of p-nitrophenyl phosphate (PNPP) and inorganic phosphate (Pi) over a large range of enzyme concentrations. From the static spectra of the pure components (ALP, PNPP, PNP, Pi), we identify their characteristic vibrational frequencies and use them as reference points for the time-resolved spectra. The reaction reveals a monotonic growth of the inorganic-phosphate band at 1077 cm−1. At the highest alkaline phosphatase concentration, we resolve two blue shifts in the nitro/aromatic region (1510 → 1518 cm−1; 1494 → 1499 cm−1), two red shifts in the fingerprint region (1345 → 1340 cm−1; 1294 → 1290 cm−1), and a splitting of the ~1592 cm−1 band into 1595 and 1583 cm−1. In conclusion, by anchoring the time-resolved spectra to the static spectra of individual constituents, we were able to resolve the infrared readout of the enzymatic reaction, offering a generalizable approach for FTIR-based tracking of catalytic processes.