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

• Published in: Biochemistry (Easton) 2000-04, Vol.39 (15), p.4259-4266
• Main Authors: Guilhaudis, Laure, Simorre, Jean-Pierre, Blackledge, Martin, Marion, Dominique, Gans, Pierre, Neuburger, Michel, Douce, Roland
• Format: Article
• Language: English
• Subjects: Amino Acid Oxidoreductases - chemistry; Amino Acid Oxidoreductases - metabolism; Apoenzymes - metabolism; Binding Sites; Biochemistry, Molecular Biology; 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; Life Sciences; Methane - analogs & derivatives; Methane - metabolism; Methylamines - metabolism; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Pisum sativum - enzymology; Polyglutamic Acid - analogs & derivatives; Polyglutamic Acid - metabolism; Protein Binding; Solutions; Tetrahydrofolates - metabolism; Thermodynamics; Thioctic Acid - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi992674w

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 CH2 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 (H4FGlu 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 H4FGlu 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 H4FGlu n greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol-1. Differences of the 1H and 15N 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.