Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate

Abstract

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.

Fig. 1

Alanine transaminase (ALT) catalytic mechanism. A proposed excursion starting at the circled “A” would explain the observed D/H exchange [–17] and is highlighted with a grey background. The inset shows the carbon nomenclature used herein.

Fig. 2

Direct ¹³C observation of ALT activity using hyperpolarized sodium pyruvate. (a) A stacked plot of individual spectra following conversion of hyperpolarized ¹³C2-pyruvate into ¹³Cα-L-alanine. The resonances corresponding to these nuclei are indicated below the horizontal axis. (b) A plot showing observed intensities extracted from the spectra shown in panel (a). (c) A stacked plot of ALT-catalyzed conversion of hyperpolarized ¹³C3D₃-pyruvate into ¹³Cβ-L-alanine. The resonances corresponding to these nuclei are indicated below the horizontal axis; note the resolution of peaks corresponding to the ¹³CD₃ (tallest peak), ¹³CD₂H, ¹³CDH₂ and ¹³CH₃ isotopologues, from right to left, respectively, for both nuclei. (d) A plot showing observed intensities extracted from the spectra shown in panel (c).

Fig. 3

Pyruvate isotopologue distributions. (a) The distribution in starting material is consistent with an incomplete deuteration reaction (~95% D). (b) The distribution observed for alanine in the first scan of an ALT reaction is different. (c) This apparent distribution changes throughout the course of an experiment, but is no longer reflective of actual chemical species because magnetization of the heavily protonated forms decays more rapidly. (d) A special experiment designed to reduce the effects of magnetization decay and observe signals from the alanine product that was produced in only 1.5 s is shown.

Fig. 4

ALT-catalyzed D/H exchange permits the indirect detection of hyperpolarized ¹³C. (a) Four alanine isotologues are produced in ¹H₂O starting from fully deuterated pyruvate. (b) This NMR pulse sequence is designed to transfer hyperpolarized ¹³C magnetization to nascent ¹H for detection and can be tuned to detect these different isotopologues. Details of this pulse sequence can be found in [10]. In panel (c), the delays denoted by τ_a in panel (b) are tuned to detect pure 13CβD2H-L-alanine (1/4J_CH = 1.96 ms).

Fig. 5

Modulating the pulse experiment on-the-fly provides insight into the product isotopologue distribution and identifies a parameter value for optimal sensitivity. (a) A single aliquot of hyperpolarized ¹³C3D₃-pyruvate was detected using the experiment in Fig. 3, except values for the delay τ_a were modulated. In the first scan the delay was 0.98 ms to detect a mixture of ¹³CD₂H- and ¹³CDH₂-alanine; likewise in the second scan with 0.66 ms. The third scan with a 1.96 ms delay will detect magnetization from only the ¹³CD₂H form, recycling the remainder. This three step acquisition pattern was repeated multiple times until the detected hyperpolarized magnetization decayed to undetectable levels. (b) Integral values of product peaks observed using the experimental parameters described in (a). The line represents a best-fit exponential decay of all plotted points.