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Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases

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Abstract

Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

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Acknowledgments

The authors thank Dr. Pablo Sabrado (Virginia Tech) for the use of Aligent 8453 UV/visible Spectrophotometer. This work is supported by NIH grant AI 19769.

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Authors and Affiliations

  1. Department of Biochemistry, Virginia Tech, Engel Hall 204, Blacksburg, VA, 24061, USA

    Pingyang Liu, Michael P. Torrens-Spence, Haizhen Ding & Jianyong Li

  2. Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Bruce M. Christensen

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  1. Pingyang Liu
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  2. Michael P. Torrens-Spence
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Correspondence to Jianyong Li.

Electronic supplementary material

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726_2012_1342_MOESM1_ESM.docx

Supplemental figure 1. Prolonged pre-incubation of cysteine with AeADC could completely inactivate the ADC activity of the enzyme. 2 μg AeADC and 1 mM of cysteine in 200 mM phosphate buffer pH=7.5 were incubated for 10 min at 25 oC prior to adding 5 mM of aspartate. The total reaction volume was 50 μl. The production of β-alanine was measured at 10 min after adding aspartate at 25 oC (DOCX 36 kb)

726_2012_1342_MOESM2_ESM.docx

Supplemental figure 2. Spectrum of supernatant from heat-denatured AeADC sample. Purified AeADC was concentrated to 10 mg per ml and treated with 2 mM of cysteine for 30 min. The treated sample was then separated by gel filtration chromatography to separate the enzyme from free cysteine. The collected protein fraction was heat-denatured and the supernatant was collected by centrifugation. The spectral characteristics of the supernatant were then analyzed using Hitachi U-2008 spectrophotometer (DOCX 88 kb)

726_2012_1342_MOESM3_ESM.docx

The model of AeADC predicted the relative positions of substrate and Q377. A. The relative position of cysteine sulfinic acid and Q377 at the active site of predicted AeADC structure. B. The relative position of aspartate and Q377 at the active site of predicted AeADC structure (DOCX 1542 kb)

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Liu, P., Torrens-Spence, M.P., Ding, H. et al. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases. Amino Acids 44, 391–404 (2013). https://doi.org/10.1007/s00726-012-1342-7

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