De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal

Jessica K. Bell, Paul J. Pease, J. Ellis Bell, Gregory A. Grant, Leonard J. Banaszak

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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 bsheet. 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 subunit-subunit 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 gradient NMR studies of the regulatory domain showed the presence of higher oligomers instead of the predicted dimer. We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.

Original languageEnglish
Pages (from-to)4176-4184
Number of pages9
JournalEuropean Journal of Biochemistry
Volume269
Issue number17
DOIs
StatePublished - Sep 25 2002

Keywords

  • 3-phosphoglycerate dehydrogenase
  • Domains
  • Enzyme regulation
  • Oxidoreductase
  • Truncation

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