IMPAIRED ASSEMBLY OF E1 DECARBOXYLASE OF THE BRANCHED-CHAIN ALPHA-KETOACID DEHYDROGENASE COMPLEX IN TYPE IA MAPLE-SYRUP-URINE-DISEASE

The E1 decarboxylase component of the human branched-chain ketoacid de hydrogenase complex comprises two E1 alpha (45.5 kDa) and two E1 beta (37.5 kDa) subunits forming an alpha(2) beta(2) tetramer, In patients with type IA maple syrup urine disease, the E1 alpha subunit is affect ed, resulting in the loss of E1 and branched-chain ketoacid dehydrogen ase catalytic activities. To study the effect of human E1 alpha missen se mutations on E1 subunit assembly, we have developed a pulse-chase l abeling protocol based on efficient expression and assembly of human ( His)(6)-E1 alpha and untagged E1 beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES, Assembly of the two S-35-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin, The nine E1 alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly wit h normal E1 beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-al pha), moderately slow (G245R-alpha), slow (G204S-alpha, A240P-alpha, F 364C-alpha, Y368C-alpha, and Y393N-alpha), and no (T265R-alpha) assemb ly. Prolonged induction in E. coli grown in the YTGK medium or lowerin g of induction temperature from 37 to 28 degrees C (in the case of T26 5R-alpha), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrif ugation showed that the wild-type E1 existed entirely as alpha(2) beta (2) tetramers, In contrast, a subset of E1 alpha missense mutations ca used the occurrence of exclusive alpha beta dimers (Y393N-alpha and F3 64C-alpha) or of both alpha(2) beta(2) tetramers and lower molecular w eight species (Y368C-alpha and T265R-alpha). Thermal denaturation at 5 0 degrees C indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min(-1)), with the T265R-alpha mut ant E1 most severely affected (rate constant, 4.45 min(-1)). The resul ts establish that the human E1 alpha mutations in the putative thiamin e pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha, have no effect on E1 subunit assembly. The T265R-alpha m utation adversely impacts both E1 alpha folding and subunit interactio ns, The mutations involving the C-terminal aromatic residues impede bo th the kinetics of subunit assembly and the formation of the native al pha(2) beta(2) structure.