Combined Structural and Biochemical Analysis of the H-T Complex in the Glycine Decarboxylase Cycle: Evidence for a Destabilization Mechanism of the H-protein

Biochemistry. 2000 Apr 18;39(15):4259-66. doi: 10.1021/bi992674w.

Abstract

The lipoate containing H-protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The transfer of the CH(2) group is catalyzed by the T-protein, which forms a 1:1 complex with the methylamine-loaded H-protein (Hmet). The methylamine group is then deaminated and transferred to the tetrahydrofolate-polyglutamate (H(4)FGlu(n)) cofactor of T-protein, forming methylenetetrahydrofolate-polyglutamate. The methylamine group is buried inside the protein structure and highly stable. Experimental data show that the H(4)FGlu(n) alone does not induce transfer of the methylene group, and molecular modeling also indicates that the reaction cannot take place without significant structural perturbations of the H-protein. We have, therefore, investigated the effect of the presence of the T-protein on the stability of Hmet. Addition of T-protein without H(4)FGlu(n) greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol(-1). Differences of the (1)H and (15)N chemical shifts of the H-protein in its isolated form and in the complex with the T-protein show that the interaction surface for the H-protein is localized on one side of the cleft where the lipoate arm is positioned. This suggests that the role of the T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Oxidoreductases / chemistry*
  • Amino Acid Oxidoreductases / metabolism*
  • Apoenzymes / metabolism
  • Binding Sites
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism*
  • Catalysis
  • Coenzymes / metabolism
  • Computer Simulation
  • Enzyme Stability
  • Formaldehyde / metabolism
  • Glycine Decarboxylase Complex
  • Glycine Decarboxylase Complex H-Protein
  • Glycine Dehydrogenase (Decarboxylating)
  • Hydrocarbons
  • Kinetics
  • Methane / analogs & derivatives
  • Methane / metabolism
  • Methylamines / metabolism
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Oxidation-Reduction
  • Peas / enzymology*
  • Polyglutamic Acid / analogs & derivatives
  • Polyglutamic Acid / metabolism
  • Protein Binding
  • Solutions
  • Tetrahydrofolates / metabolism
  • Thermodynamics
  • Thioctic Acid / metabolism

Substances

  • Apoenzymes
  • Carrier Proteins
  • Coenzymes
  • Glycine Decarboxylase Complex H-Protein
  • Hydrocarbons
  • Methylamines
  • Solutions
  • Tetrahydrofolates
  • tetrahydrofolate polyglutamate
  • Formaldehyde
  • carbene
  • Polyglutamic Acid
  • Thioctic Acid
  • 5,10-methylenetetrahydrofolate polyglutamate
  • methylamine
  • Amino Acid Oxidoreductases
  • Glycine Decarboxylase Complex
  • Glycine Dehydrogenase (Decarboxylating)
  • Methane