Detection of Reaction Intermediates during Human Cystathionine beta-Synthase-monitored Turnover and H2S Production

作者：Yadav Pramod Kumar; Banerjee Ruma^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287(52): 43464-43471.

DOI：10.1074/jbc.M112.414722

Human cystathionine beta-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H2O or H2S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5'-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of L-serine and L-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k(obs) = 0.96 +/- 0.02 and 0.38 +/- 0.01 mM(-1) s(-1), respectively, at 24 degrees C) with concomitant loss of H2O and H2S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k(obs) = 40.6 +/- 3.8 s(-1) and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k(obs) = 0.38 +/- 0.01 mM(-1) s(-1). This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.

• 出版日期2012-12-21