Abstract：
:Cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals, i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H(2)S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457-22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H(2)S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H(2)S generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H(2)S and forms an aminoacrylate intermediate (k(obs) of 1.61 ± 0.04 mM(-1) s(-1) at low cysteine concentrations and 2.8 ± 0.1 mM(-1) s(-1) at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM(-1) s(-1) and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products, cystathionine and H(2)S.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Singh S,Ballou DP,Banerjee Rdoi
10.1021/bi1010893subject
Has Abstractpub_date
2011-01-25 00:00:00pages
419-25issue
3eissn
0006-2960issn
1520-4995journal_volume
50pub_type
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