Abstract
Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H(2)O or H(2)S, 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 °C) with concomitant loss of H(2)O and H(2)S 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.
Highlights
Human cystathionine -synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to give cystathionine and H2O or H2S
We employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions
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
Summary
Human cystathionine -synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to give cystathionine and H2O or H2S. Human cystathionine -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. 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. In the second half-reaction, the aminoacrylate intermediate undergoes nucleophilic attack by the thiolate of homocysteine to yield the external aldimine of the product, cystathionine. We used difference electronic absorption spectroscopy to monitor the reaction kinetics of full-length human CBS under pre-steady-state conditions. The reactions catalyzed in the presence of serine or cysteine provide, for the first time, kinetic insights into the canonical transsulfuration reaction and the H2S-producing reactions catalyzed by human CBS
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