Measurement of Kinetics and Active Site Distances in Metalloenzymes Using Paramagnetic NMR with C-13 Hyperpolarization

Abstract

Paramagnetic relaxation enhancement (PRE) conjoint with hyper polarized NMR reveals structural information on the enzyme-product complex in an ongoing metalloenzyme-catalyzed reaction. Substrates of pseudouridine mono phosphate glycosidase are hyperpolarized using the dynamic nuclear polarization (DNP) method. Time series of C-13 NMR spectra are subsequently measured with the enzyme containing diamagnetic Mg2+ or paramagnetic Mn2+ ions in the active site. The differences of the signal evolution and line widths in the Mg2+ vs Mn2+ reactions are explained through PRE in the enzyme-bound product, which is in fast exchange with its free form. Here, a strong distance dependence of the paramagnetically enhanced relaxation rates enables the calculation of distances from product atoms to the metal center in the complexed structure. The same method can be used to add structural information to real-time characterizations of chemical processes involving compounds with naturally present or artificially introduced paramagnetic sites.