Active site mutants of pig citrate synthase (PCS)

Claudia T. Evans, Linda C. Kurz, S. James Remington, P. Srere

Research output: Contribution to journalArticle

Abstract

Several changes were made at specific residues in the PCS active site to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. Most mutations affected the kcat and Km for OAA and AcCoA and included: H274G, H274R, D375G, D375N, D375E, D37SQ, H320G, H320Q, H320N, H320R, R401H, G275V and G275A There was only one mutant, D327N, in which the Km primarily was affected. The mutations, R401G, R401K, H235Q and N242E, had smaller effects on kcat and Km. R401K exhibited significantly improved kcat/Km The biomolecular basis for altered fonction was assessed by comparing the AAG changes of kcat/Km X-ray crystallographic studies at 2.7A resolution of one of the mutants, H274G, revealed that the mutant enzyme crystallizes in an "open" conformation with a localized relaxation of the structure to relieve the strain imposed by a high energy main and side chain conformation of H274 in the non-mutant. Electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the PCS catalyzed reaction may be an "enolized" version of AcCoA that is neither neutral nor fully negatively charge and that a possible role for the catalytically essential histidines is to stabilize the intermedicate by charge delocalization.

Original languageEnglish
JournalFASEB Journal
Volume10
Issue number6
Publication statusPublished - 1996

Fingerprint

Citrate (si)-Synthase
citrate (si)-synthase
active sites
Conformations
Catalytic Domain
Swine
mutants
Mutation
swine
Enzymes
Hydrogen Bonding
Static Electricity
Histidine
mutation
Electrostatics
Hydrogen bonds
enzyme kinetics
hydrogen bonding
X-Rays
histidine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Evans, C. T., Kurz, L. C., James Remington, S., & Srere, P. (1996). Active site mutants of pig citrate synthase (PCS). FASEB Journal, 10(6).

Active site mutants of pig citrate synthase (PCS). / Evans, Claudia T.; Kurz, Linda C.; James Remington, S.; Srere, P.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

Evans, CT, Kurz, LC, James Remington, S & Srere, P 1996, 'Active site mutants of pig citrate synthase (PCS)', FASEB Journal, vol. 10, no. 6.
Evans CT, Kurz LC, James Remington S, Srere P. Active site mutants of pig citrate synthase (PCS). FASEB Journal. 1996;10(6).
Evans, Claudia T. ; Kurz, Linda C. ; James Remington, S. ; Srere, P. / Active site mutants of pig citrate synthase (PCS). In: FASEB Journal. 1996 ; Vol. 10, No. 6.
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