Toward a General Method to Observe the Phosphate Groups of Phosphoenzymes with Infrared Spectroscopy

Abstract

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.

Conformational changes of recombinant Ca2+–ATPase studied by reaction‐induced infrared difference spectroscopy

“…This indicates that only~50% of the protein in the FTIR spectra of formation of E2P Figure 4 shows difference spectra for the reaction from Ca 2 E1 to E2P in the presence of 10% Me 2 SO and absence of K + [27]. These conditions accumulate protein in the E2P state after ATP release, which is indicated by the amplitude of the bands at 1689 and 1607 cm À1 [23,26,32,33]. Another marker band for the E2P state is found near 1193 cm À1 , which has been assigned to the phosphate group by isotopic labeling experiments [33].…”

Microhydrated dihydrogen phosphate clusters probed by gas phase vibrational spectroscopy and first principles calculations

“…Hydrolysis of phosphates also produces phosphate ions as intermediate and biochemical signals, 5 which can then be monitored using vibrational spectroscopy in reaction kinetics studies. [6][7][8][9][10] Understanding the solvation dynamics of hydrated phosphate ions by computer simulations 1,[11][12][13][14][15][16][17] is of fundamental importance, as it provides the basis for the interpretation of spectroscopic data. [18][19][20][21][22][23][24] These simulations require a detailed knowledge of the interaction between phosphate ions and water.…”

Vibrational Spectra of Phosphate Ions in Aqueous Solution Probed by First-Principles Molecular Dynamics

“…Vibrational spectroscopy is a versatile technique to monitor such reactions. − If one could decode such spectra in terms of the underlying molecular structures and interactions, one additionally might be able to pin down the detailed mechanisms of these reactions. Hybrid methods, which combine a density functional theory (DFT) treatment of a molecule with a simplified molecular mechanics (MM) description of its condensed-phase environment, should be well-suited for such a theoretical analysis .…”