A continuous spectrophotometric assay for human cystathionine beta-synthase

Weijun Shen, Molly K. McGath, Ruby Evande, David B. Berkowitz

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

We report a new continuous spectrophotometric assay for human cystathionine beta-synthase (hCBS). This assay relies upon the finding that hCBS will take cysteamine in place of l-homocysteine, thereby producing thialysine. Thialysine is, in turn, decarboxylated by lysine decarboxylase, releasing CO2 that is monitored by the sequential action of phosphoenolpyruvate carboxylase and l-malate dehydrogenase. The decrease in absorbance at 340 nm is monitored as reduced nicotinamide adenine dinucleotide is consumed. Using this four-enzyme couple, we find that Kmapp=1.2±0.2mM for l-serine and 5.6 ± 2.2 mM for cysteamine, with kcat = 1.3 ± 0.1 s-1 for the formation of thialysine by hCBS. For comparison purposes, the same hCBS reaction was monitored via a radioactive single time point assay using 14C-(C-1)-labeled l-serine and cysteamine as substrates, counting the thialysine product, following ion exchange chromatography. This assay yielded Kmapp=2.2±0.5mM for l-serine and 6.6 ± 2.2 for cysteamine, with kcat = 2.5 ± 0.4 s-1. These numbers indicate that, although it possesses a shortened carbon chain and lacks a carboxyl group, cysteamine displays a catalytic efficiency (k cat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, l-homocysteine.

Original languageEnglish (US)
Pages (from-to)103-110
Number of pages8
JournalAnalytical Biochemistry
Volume342
Issue number1
DOIs
StatePublished - Jul 1 2005

Keywords

  • Continuous assay
  • Cystathionine beta-synthase
  • Cysteamine
  • Lysine decarboxylase
  • Malate dehydrogenase
  • Phosphoenolpyruvate carboxylase
  • Thialysine

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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