We propose to develop a practical and convenient method to quantitatively characterize the accessibility and electronic stat of metalloprotein active sites. The method, which we call PhoRE (for Phosphorus Relaxation Enhancement) relies on correlating changes in the 31 P NMR line widths of chemical probe molecules with the rate at which the probes come into physical contact with paramagnetetic ion(s) at the enzyme~s active site. Their chemical and magnetic properties make dialkylphosphate probes uniquely suited for this application. While this manuscript describes a study of the inhibition and regulation of protein phosphatase 1 (PP1), the method does not require that the chemical structure of the probe resemble that of the enzyme~s substrate. We stress that the greater understanding of enzyme regulation and disease mechanisms, and to new methods for screening drug candidates.
The specific aims of the study are: (1) to characterize differences in the availability of the active sites of different stable conformations of PP1 and determine the effects of natural toxins, small ionic inhibitors, and regulatory elements on active site availability. We will correlate these differences with known changes in enzymatic activity. (2) We will measure the sensitivities of these interactions to changes in the steric bulk, electronic charge, and nucleophilicity of the probe molecules, thereby correlating changes in reactivity with local conformational changes at the active site. (3) We will further characterize the method by studying interactions of the probes with structurally characterized synthetic metal complexes, and with myoglobin, and the manganese and iron superoxide dismutases. (4) Our long term objective is to apply the method to a wide variety of metalloproteins. We will compare the activities of structurally similar metalloenzyme and correlate differences in activities with structural differences. This method has the potential to provide chemical explanations for changes in enzyme behavior that accompany changes in conformations associated with binding inhibitors, toxins, and regulatory elements, and with disease causing mutations..
| Summers, Jack S; Base, Karel; Boukhalfa, Hakim et al. (2005) Use of phosphorus ligand NMR probes to investigate electronic and second-sphere solvent effects in ligand substitution reactions at manganese(II) and manganese(III). Inorg Chem 44:3405-11 |
| Summers, J S; Hoogstraten, C G; Britt, R D et al. (2001) 31P NMR probes of chemical dynamics: paramagnetic relaxation enhancement of the (1)H and (31)P NMR resonances of methyl phosphite and methylethyl phosphate anions by selected metal complexes. Inorg Chem 40:6547-54 |
