Elucidating the Heme Domain's Regulatory Role on Human Cystathionine β‐Synthase (hCBS) Product Formation

Abstract

Human cystathionine β‐synthase (hCBS) is a vital enzyme found within the human transulfuration pathway and is responsible for the biotransformation of serine and homocysteine into cystathionine. hCBS at high cysteine concentrations has been shown to release hydrogen sulfide as a byproduct via an alternate reaction, suggesting hCBS to be closely involved with regulating metabolic sulfur. Catalysis in both the primary and alternate reactions proceeds via a PLP dependent β‐replacement ping‐pong mechanism, with the last step before product dissociation from the internal aldimine (Schiff base) intermediate being the condensation of H2O or H2S, respectively. Consequently, mis‐regulation of substrate levels or loss of CBS activity has been causally linked to hyperhomocysteinemia, a biological condition signifying abnormally high levels of homocysteine within the circulatory system; this condition being one of many causes leading to cardiovascular disease. A highly conserved PLP catalytic and SAM regulatory domain can be seen when comparing yeast analogue (yCBS) to mammalian variants. However, the mammalian variants, specifically the human analog hCBS, differs by containing a unique heme binding domain not found in yCBS. Given that the heme prosthetic group is not required for catalytic activity, the role that this additional domain plays in regulating cystathionine production in hCBS is not yet clearly understood. Current methods for measuring CBS catalytic activity involve using radioactively labeled substrates and typically have focused only on yeast and truncated hCBS variants. We have adapted a previously established colorimetric coupled enzyme assay to measure hCBS kinetics, coupling CBS with cystathionine β‐lyase (CBL), and lactate dehydrogenase (LDH); where the stoichiometric oxidation of NADH, monitored spectrophotometrically at 340 nm, is correlated directly to the formation of cystathionine. Preliminary results suggest this coupled assay is a viable method for measuring product formation kinetics of full length wild type hCBS. Current and future work aims to 1) asses catalytic rate activity for wild type hCBS and 2) asses the activity of single point mutant variants with mutations localized within the heme binding domain. Through these experiments, we hope to elucidate a deeper understanding of how the heme binding domain affects cystathionine product formation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.