Structural and functional characterization of H protein mutants of the glycine decarboxylase complex

The mitochondrial glycine decarboxylase complex (GDC) consists of four comp onent enzymes (P, H, T, and L proteins) involved in the breakdown of glycin e. in order to investigate structural interactions involved in the stabiliz ation of the methylamine-loaded H protein (a transient species in the GDC r eaction), we designed several mutants of H apoprotein. Structural analysis of the wild-type and mutants of H apoprotein emphasized the necessity to carefully assess, by biophysical techniques, the correct folding of mutated proteins prior to investigate their biochemical properti es. The correctly folded wild-type and mutants of H apoprotein were in vitr o lipoylated and then characterized in the context of GDC reaction by study ing the reconstituted complex and partial reactions. We showed that Val(62) and Ala(64), surrounding the lipoyl-lysine, play an important role in the molecular events that govern the reaction between P and H protein but do no t intervene in the recognition of the binding site of Lipoic acid by lipoyl ligase, The biochemical results obtained with the HE14A mutant of H protei n pointed out the major role of the Glu(14) amino acid residue in the GDC c atalysis and highlighted the importance of the ionic and hydrogen bounds in the hydrophobic cleft of H protein for the stabilization of the methylamin e-loaded lipoyl arm.