On the mechanism of pyridoxine responsive homocystinuria. II. Properties of normal and mutant cystathionine beta-synthase from cultured fibroblasts.

Cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] was studied in cultured skin fibroblasts from two control subjects and three patients with pyridoxine-responsive homocystinuria. In crude cell sonicates, cystathionine synthase activity detected in each mutant line was less than 5% of control values. After differential centrifugation, ammonium sulfate fractionation, and calcium phosphate gel treatment, the specific activity of synthase from control lines increased 5- to 7-fold with 70-79% yield. These same steps led to only 2- to 3-fold purification of mutant synthase and a reduced yield (26-44%). Michaelis-Menten analyses with the partially purified enzyme revealed that each mutant synthase had a marked reduction in affinity for its coenzyme, pyridoxal 5'-phosphate, as well as reduced affinity and maximum velocity for both co-substrates, L-homocysteine and L-serine. Even at saturating concentrations of coenzyme, mutant synthase activity was less than 3% of control. Mutant synthase was also far more thermolabile than control enzyme. In the absence of added coenzyme, heating for 10 min at 55 degrees led to complete loss of mutant activity whereas control activity was reduced by 60%. Significantly, addition of saturating concentration of coenzyme prior to heating increased thermostability of both control and mutant synthase, the fractional increase being considerably greater in the mutants. We conclude that these patients suffer from a mutation of the synthase apoenzyme which impairs coenzyme binding, and that this primary abnormality results in reduced total enzyme activity in two ways: by reducing holoenzyme formation; and by accelerating apoenzyme degradation. We propose that pharmacologic amounts of pyridoxine increase holoenzyme formation modestly, thereby enhancing catalytic activity and slowing apoenzyme turnover.