Article

A continuous spectrophotometric assay for human cystathionine beta-synthase

Department of Biochemistry, University of Nebraska at Lincoln, Lincoln, Nebraska, United States
Analytical Biochemistry (Impact Factor: 2.22). 08/2005; 342(1):103-10. DOI: 10.1016/j.ab.2005.03.051
Source: PubMed

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 Km(app) = 1.2+/-0.2 mM for L-serine and 5.6+/-2.2 mM for cysteamine, with kcat = 1.3+/-0.1s(-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 Km(app) = 2.2+/-0.5 mM 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 (kcat/Km) with hCBS that is within an order of magnitude of that observed with its natural thiol cosubstrate, L-homocysteine.

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