Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine beta-synthase.

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Taoka S, Banerjee R

Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine beta-synthase.

J Biol Chem. 2002 Jun 21;277(25):22421-5. Epub 2002 Apr 10.

PubMed ID

11948191 [ View in PubMed

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Abstract

Cystathionine beta-synthase found in yeast catalyzes a pyridoxal phosphate-dependent condensation of homocysteine and serine to form cystathionine. Unlike the homologous mammalian enzymes, yeast cystathionine beta-synthase lacks a second cofactor, heme, which facilitates detailed kinetic studies of the enzyme because the different pyridoxal phosphate-bound intermediates can be followed by their characteristic absorption spectra. We conducted a rapid reaction kinetic analysis of the full-length yeast enzyme in the forward and reverse directions. In the forward direction, we observed formation of the external aldimine of serine (14 mm(-1) s(-1)) and the aminoacrylate intermediate (15 s(-1)). Homocysteine binds to the aminoacrylate with a bimolecular rate constant of 35 mm(-1) s(-1) and rapidly converts to cystathionine (180 s(-1)), leading to the accumulation of a 420 nm absorbing species, which has been assigned as the external aldimine of cystathionine. Release of cystathionine is slow (k = 2.3 s(-1)), which is similar to k(cat) (1.7 s(-1)) at 15 degrees C, consistent with this being a rate-determining step. In the reverse direction, cystathionine binds to the enzyme with a bimolecular rate constant of 1.5 mm(-1) s(-1) and is rapidly converted to the aminoacrylate without accumulation of the external aldimine. The kinetic behavior of the full-length enzyme shows notable differences from that reported for a truncated form of the enzyme lacking the C-terminal third of the protein (Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) Biochemistry 40, 10873-10880).

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Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Pyridoxal phosphate	Cystathionine beta-synthase	Protein	Humans	Unknown	Cofactor	Details