A continuous spectrophotometric assay for human cystathionine beta-synthase.
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.
Shen
Weijun
W
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
McGath
Molly K
MK
Evande
Ruby
R
Berkowitz
David B
DB
D002262:Carboxy-Lyases / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000032:analysis*; D003543:Cysteamine / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
C000204:S-2-aminoethyl cysteine; D003543:Cysteamine; D008291:Malate Dehydrogenase; D002262:Carboxy-Lyases; C021520:lysine decarboxylase; D010730:Phosphoenolpyruvate Carboxylase; D003541:Cystathionine beta-Synthase; D003545:Cysteine