EXPRESSION AND KINETIC CHARACTERIZATION OF METHYLMALONYL-COA MUTASE FROM PATIENTS WITH THE MUT- PHENOTYPE - EVIDENCE FOR NATURALLY-OCCURRING INTERALLELIC COMPLEMENTATION

L-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requ iring mitochondrial matrix enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Inherited defects in the gene enc oding this enzyme result in the mut forms of methylmalonic acidemia, E xpression of mature human MUT cDNA in Escherichia coil at a post-induc tion cultivation temperature of 12 degrees C, rather than 37 degrees C , led to the folding of the majority of the synthesized protein to a s oluble form, with an activity of 0.2-0.3 U/mg protein in the cell-free extract, 10-15 times higher than that in human liver homogenate, Six missense mutations, producing the amino acid changes G94V, Y231N, R369 H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffe ring from the mut(-) form of methylmalonic acidemia, resulting from de fective AdoCbl binding, Two (G623R and G717V) had been reported in oth er patients, Three (G94V, Y231N and R369H) are the first changes in th e NH2-terminal part of the enzyme reported to cause the mut(-) phenoty pe, Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients. The mutations increased the K-m for AdoCbl by 40- to 900-fold, while V-max values va ried from 0.2% to nearly 100% of that of wild-type protein, In one cas e of a doubly heterozygous cell line, however, neither of the constitu ent mutant enzymes had a K-m corresponding to the lower of the two est imated from the extract data, This finding may reflect the natural occ urrence of interallelic complementation in vivo in this cell line.