کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533184 | 961855 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A continuous spectrophotometric assay for human cystathionine beta-synthase
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
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 sâ1 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 sâ1. 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 103-110
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 103-110
نویسندگان
Weijun Shen, Molly K. McGath, Ruby Evande, David B. Berkowitz,