We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut° mutants. Those with detectable residual activity in cell extracts (∼0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut− mutants. The mutant enzyme in the mut− mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (Km) for adenosylcobalamin in vitro, a normal Km for methylmalonyl CoA, and a strikingly reduced thermal stability at 45°C relative to control. Mutase from one mut− mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.
To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut° or mut− mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut− mutants exhibited complex enzyme kinetics; ∼80% of mutase activity demonstrated a Km indistinguishable from control, whereas a smaller component of activity exhibited a Km similar to the abnormal Km expressed by the mut− propositus in each family. In two families with a mut° propositus, mutase from three of the four parents exhibited only the normal Km for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut+) and a mutant allele with an abnormal Km. From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut°, mut−, and mut+ alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut° or mut− alleles from their parents.