Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate.
Hyperpolarized metabolites offer a tremendous sensitivity advantage (>10(4) 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, (13)C-enriched analog of pyruvic acid, (13)C3D(3)-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct (13)C observation and indirect observation through methyl protons introduced by ALT-catalyzed H-D exchange. Exchange on injecting hyperpolarized (13)C3D(3)-pyruvate into ALT dissolved in buffered (1)H(2)O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5s 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 (13)C3-pyruvate and products in imaging applications are discussed.
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, United States.
A W
AW
Barb
S K
SK
Hekmatyar
J N
JN
Glushka
J H
JH
Prestegard
D000410:Alanine Transaminase / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted
D002247:Carbon Isotopes; D011522:Protons; D019289:Pyruvic Acid; D003903:Deuterium; D000410:Alanine Transaminase