A Pathogenic Linked Mutation in the Catalytic Core of Human Cystathionine β-synthase Disrupts Allosteric Regulation and Allows Kinetic Characterization of a Full-length Dimer

摘要

Cystathionine β-synthase catalyzes the condensation of serine and homocysteine to yield cystathionine and is the single most common locus of mutations associated with homocystinuria. In this study, we have examined the kinetic consequences of a pair of linked patient mutations, P78R/K102N, that are housed in the catalytic core of the protein and compared it to the effects of the corresponding single mutations. The P78R mutation affords purification of a mixture of higher order oligomers, P78R-I, which resembles the mixed quaternary state associated with wild type enzyme. However, unlike wild type enzyme, P78R-I converts over time to P78R-II, which exists predominantly, as a full-length dimer. The specific activities of the K102N, P78R-I and P78R-II mutants in the absence of AdoMet are ~3-, 9- and 3-fold lower than of wild-type enzyme and are stimulated 2.9-, 2.5- and 1.4-fold respectively by AdoMet. However, when linked, the specific activity of the resulting double mutant is comparable to that of wild-type enzyme but it is unresponsive to AdoMet, revealing that interactions between the two sites modulate the phenotype of the enzyme. Steady-state kinetic analysis for the double mutant reveals a sigmoidal dependence on homocysteine that is not observed with wild-type enzyme, which is ascribed to the mutation at the K102 locus and indicates changes in subunit interactions. Hydrogen-deuterium mass spectrometric analyses reveals that even in the absence of AdoMet, the double mutant is locked in an activated conformation that is observed for wild-type enzyme in the presence of AdoMet, providing a structural rationale for loss of this allosteric regulation. To our knowledge, this is the first example of mutations in the catalytic core of cystathionine β-synthase that result in failure of AdoMet-dependent regulation. Furthermore, analysis of individual single mutations has permitted for the first time, partial kinetic characterization of a full-length dimeric form of human cystathionine β-synthase.