Citrate lyase deacetylase of Rhodopseudomonas gelatinosa. Isolation of the enzyme and studies on the inhibition by L-glutamate

Eur J Biochem. 1980 Oct;111(2):461-71. doi: 10.1111/j.1432-1033.1980.tb04961.x.

Abstract

Citrate lyase deacetylase or acetyl-S-(acyl-carrier protein) enzyme thioester hydrolase (acetate) (EC 3.1.2-), was purified 3100-fold with a yield of 3.8% from cell extracts of Rhodopseudomonas gelatinosa. The final enzyme preparation gave a single protein band upon polyacrylamide-gel electrophoresis in the absence or in the presence of sodium dodecylsulfate. The molecular weight of the native enzyme was estimated by gel filtration to be 14 300 +/- 1000. Sodium dodecylsulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 7300 +/- 600 indicating that the enzyme consisted of two subunits. Citrate lyase deacetylase acted as an S-acetyl enzyme thioesterhydrolase because it catalyzed the conversion of citrate lyase (S-acetyl form) into citrate lyase (sulfhydryl form) and acetate. Citrate lyase deacetylase was strongly inhibited by L-glutamate. The half-maximal inhibitor concentration was 7.5 X 10(-4) M. and a Ki-value of 1.2 X 10(-4) M was determined. The mode of inhibition appeared to be of the linear mixed type. L-Glutamate was bound by citrate lyase deacetylase but not by citrate lyase. The pool concentrations of L-glutamate in R. gelatinosa were 10 mM when citrate was present in substrate amounts and 2.7 mM after total consumption of citrate. Simulation of conditions in vivo using homogeneous enzyme preparations of citrate lyase and citrate lyase deacetylase, and glutamate concentrations of 10 mM and 2.7 mM respectively, revealed that the rate of citrate lyase inactivation increased from 8% within 15 min during growth on citrate to 50% within 15 min in the absence of citrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Glutamates / pharmacology*
  • Kinetics
  • Ligands
  • Molecular Weight
  • Oxo-Acid-Lyases / antagonists & inhibitors
  • Oxo-Acid-Lyases / isolation & purification*
  • Protein Binding
  • Rhodopseudomonas / enzymology*
  • Structure-Activity Relationship
  • Thiolester Hydrolases / antagonists & inhibitors
  • Thiolester Hydrolases / isolation & purification*

Substances

  • Glutamates
  • Ligands
  • Thiolester Hydrolases
  • citrate (pro-3S)-lyase deacetylase
  • Oxo-Acid-Lyases