Inherited Methylmalonyl CoA Mutase Apoenzyme Deficiency in Human Fibroblasts: EVIDENCE FOR ALLELIC HETEROGENEITY, GENETIC COMPOUNDS, AND CODOMINANT EXPRESSION

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Abstract

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.

Authors

Huntington F. Willard, Leon E. Rosenberg