Subtle Defects of Four Homocystinuric Variants of Human Cystathionine β‐Synthase

In humans, CBS‐deficient homocystinuria (CBSDH) results from mutations in the gene encoding cystathionine β‐synthase (CBS), a heme‐containing PLP‐dependent enzyme that catalyzes condensation of serine and homocysteine to form cystathionine. The role of the heme in CBS is unknown; however, disruption of the native heme iron‐to‐cysteine thiolate bond eliminates enzyme activity even though the heme does not participate in catalysis. Four CBSDH‐associated single amino acid variants within the heme pocket, P49L, P78R, R125Q and R266K, were investigated through studies of their heme environments and catalytic properties. Electronic absorption, resonance Raman, and electron paramagnetic resonance spectroscopic studies revealed that there are subtle changes to the heme environment in these variants, consistent with changes in hydrogen bonding or local heme pocket structure. Most variants exhibited a less stable heme pocket, susceptible to thermal unfolding at lower temperatures than for the wild‐type protein. Variant proteins were often more stable to thermally mediated aggregation resulting from global protein unfolding. Kinetic characterization revealed that the allosteric activator S ‐adenosylmethionine (AdoMet) lowered K m for both substrates but had little effect on k cat for the wild‐type protein, a result consistent with the current structural model for AdoMet activation. P49L, P78R and R266K exhibited subtle changes in their kinetic constants and were AdoMet responsive. In contrast, R125Q exhibited a significantly decreased k cat value and was unresponsive to AdoMet activation. The correlation between altered heme environment and catalytic activity suggests that the heme plays an important regulatory role in this protein. Support or Funding Information University of Wisconsin‐Madison