TAILIEUCHUNG - Báo cáo khoa học: De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal

Escherichia coli 3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibitedby serine. Structural studies revealeda homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains: nucleotide, substrate and regu-latory. InPGDH, extensive interactions are formedbetween nucleotide binding domains. | Eur. J. Biochem. 269 4176-4184 2002 FEBS 2002 doi De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal Jessica K. Bell1 Paul J Pease1 J Ellis Bell2 Greaorv A Grant3 and Leonard J Banaszak1 1 Department of Biochemistry Molecular Biology and Biophysics University of Minnesota Minneapolis MN USA 2Department of Chemistry University of Richmond Richmond Virginia USA 3Department of Molecular Biology and Pharmacology and the Department of Medicine Washington University St Louis MO USA Escherichia coli 3-phosphoglycerate dehydrogenase PGDH catalyzes the first step in serine biosynthesis and is allosterically inhibited by serine. Structural studies revealed a homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains nucleotide substrate and regulatory. In PGDH extensive interactions are formed between nucleotide binding domains. A second subunit-subunit interaction occurs between regulatory domains creating an extended b sheet. The serine-binding sites overlap this interface. In these studies the nucleotide and substrate domains NSDs were subcloned to identify changes in both catalytic and physical properties upon removal of a subunitsubunit interface. The NSDs did not vary significantly from PGDH with respect to kinetic parameters with the exception that serine no longer had an effect on catalysis. Temperature dependent dynamic light scattering DLS revealed the NSDs aggregated 5 C before PGDH indicating decreased stability. DLS and gel filtration studies showed that the truncated enzyme formed a tetramer. This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated closely related dimeric enzymes. Expression of the regulatory domain to study conformational changes induced by serine binding yielded a product that by CD spectra contained stable secondary structure. DLS and pulsed field .

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