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

ABSTRACT: Paramagnetic relaxation enhancement (PRE) conjoint with hyperpolarized NMR reveals structural information on the enzyme-product complex in an ongoing metalloenzyme-catalyzed reaction. Substrates of pseudouridine monophosphate glycosidase are hyperpolarized using the dynamic nuclear polarization (DNP) method. Time series of ¹³C 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 calc

SUBMITTER: Liu M 

PROVIDER: S-EPMC6200339 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature