Deletion of the regulatory domain in the pyridoxal phosphate-dependent heine protein cystathionine β-synthase alleviates the defect observed in a catalytic site mutant

Abstract

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine β-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B6-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The ~13-fold diminution in activity because of the mutation corresponds to an ~7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine β-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B6-responsive phenotype associated with a cystathionine β-synthase-impaired homocystinuric genotype.