Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate

摘要

Hyperpolarized metabolites offer a tremendous sensitivity advantage (>10⁴ fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, ¹³C-enriched analog of pyruvic acid, ¹³C3D3-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct ¹³C observation and indirect observation through methyl protons introduced by ALT-catalyzed H–D exchange. Exchange on injecting hyperpolarized ¹³C3D3-pyruvate into ALT dissolved in buffered ¹H2O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5 s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized ¹³C3-pyruvate and products in imaging applications are discussed.