Interactions of methylmalonyl CoA mutase from normal human fibroblasts with adenosylcobalamin and methylmalonyl CoA: evidence for non-equivalent active sites

We have examined interactions between human methylmalonyl CoA mutase and two critical ligands, its cofactor adenosylcobalamin (AdoCbl) and its substrate methylmalonyl CoA, by performing in vitro experiments with preparations of mutase apoenzyme and holoenzyme from normal cultured human fibroblasts. When extracts are prepared from cells grown in medium containing high concentrations of hydroxocobalamin, a precursor of AdoCbl, mutase activity measured in Tris-containing buffers in the absence of added AdoCbl accounts maximally for only 50% of that activity measured in the presence of excess AdoCbl. A similar result is observed when mutase holoenzyme is formed in vitro by incubating cell extracts containing apoenzyme with AdoCbl and removing excess AdoCbl by gel filtration. When such holoenzyme preparations are heated at 45 °C and then assayed for activity, their thermostability is less than that of mutase holoenzyme heated in the presence of excess cofactor, but far greater than that of mutase apoenzyme. Methylmalonyl CoA modulates these enzyme-coenzyme interactions, since mutase holoenzyme formed in Triscontaining buffers is resolved to apoenzyme upon exposure to substrate. Qualitatively different data are obtained when buffers containing cations other than Tris are used. Under these conditions, mutase activity measured in the absence of added AdoCbl accounts for nearly 100% of the activity measured in the presence of excess cofactor, whether holoenzyme is formed in intact cells in culture or in cell extracts in vitro. Furthermore, holoenzyme formed in vitro in potassium phosphate buffer is not resolved to apoenzyme upon exposure to substrate. We suggest that the “holoenzyme” form of mutase obtained and assayed in Tris-containing buffers is that molecular species with only one of its two potential AdoCbl binding sites occupied in a catalytically active fashion, and that other ions can influence markedly the interactions between mutase, AdoCbl, and methylmalonyl CoA. These data are consistent, therefore, with the hypothesis that the dimeric mutase apoenzyme is characterized, under certain conditions, by nonequivalent active sites.