31P NMR Studies of the Complex of NADPH and Methotrexate with Lactobacillus Casei Dihydrofolate Reductase in the Solid State-Comparisons with Studies in Solution

Abstract

High resolution solid state 31P NMR spectroscopy has been utilized for studying complexes of the coenzyme NADPH (Structure 1) and methotrexate (MTX) with Lactobacillus casei dihydrofolate reductase (DHFR) using the techniques of cross polarization, magic angle spinning and high power proton decoupling [1,2]. Isotropic chemical shifts show similar trends to those measured in solution [3], The principal components of the shielding tensors and asymmetry parameters for the 31P nuclei in the 2’-phosphate and pyrophosphate groups have been determined in the lyophilized powder and on low level hydrated samples. The shielding tensor elements were found to depend strongly on the ligand ionization. The spectra of NADPH bound to lyophilized enzyme indicate the presence of two ionization states of the 2’-phosphate group corresponding to the monoanionic and dianionic forms (Figure 1). Low level hydration has a profound effect on the spectral resolution and on the ionization state of the bound NADPH [2]. Magnetization transfer [4] between protons of highly mobile water molecules and NADPH bound to hydrated, at low level, DHFR has been achieved [2]. 31P NMR spectroscopy in the solid state should prove very useful for studying protein complexes of phosphorus containing coenzymes.