Watching Alkaline Phosphatase Catalysis Through Its Vibrational Fingerprint.

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.