期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 281, 期 19, 页码 13433-13438出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M600246200
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资金
- NHLBI NIH HHS [HL58984, R01 HL058984, R01 HL058984-08] Funding Source: Medline
- NIGMS NIH HHS [R01 GM033576, R01 GM033576-40, GM33576] Funding Source: Medline
Cystathionine beta- synthase ( CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate- dependent reaction. It also contains a heme cofactor to which carbon monoxide ( CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 mu M-1. Stopped flow measurements reveal slow CO association ( 0.0166 s (-1)) limited by dissociation of the endogenous ligand, Cys- 52. Rebinding of CO and of Cys- 52 following CO photodissociation were independently monitored via time- resolved resonance Raman spectroscopy. The Cys- 52 rebinding rate, 4000 s(-1), is essentially unchanged between pH7.6 and 10.5, indicating that the pK(a) of Cys- 52 is shifted below pH7.6. This effect is attributed to the nearby Arg- 266 residue, which is proposed to form a salt bridge with the dissociated Cys- 52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg- 266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.
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