| This work explored the function of heme in cystathionine beta-synthase (CBS) by studying the interplay of heme reactivities and enzymatic activities in CBS orthologs and mutants. CBS is a pyridoxal 5'-phosphate (PLP)-dependent enzyme involved in sulfur amino acid metabolism. CBS catalyzes the condensation of serine and homocysteine to form cystathionine via the PLP cofactor. Missense mutations in CBS are associated with the hereditary disease CBS-deficient homocystinuria (CBSDH). Human CBS is regulated by S-adenosylmethionine (AdoMet) and unexpectedly contains heme, whose function remains elusive. The His/Cys coordination environment of the CBS heme confers unusual reactivities. Disruption of the heme-Cys(thiolate) coordinate bond (e.g. by binding CO or NO gas to the heme) eliminates enzyme activity. A comparative study of human and fruit fly CBSs revealed that the fruit fly CBS is highly active but is not regulated by AdoMet due to its inability to bind AdoMet. The fruit fly CBS contains a heme which behaves essentially the same as the human CBS heme. These results suggest that the heme's function is not related to AdoMet-regulation. The CBS heme binds CO and NO extremely slowly and is therefore unlikely to be a gas sensor. In the CBSDH-associated variants P49L, P78R, R125Q and R266K, the heme environment is disturbed, as indicated by subtle changes in the spectroscopic properties of the variants. The P49L, P78R and R266K variants are catalytically competent and AdoMet-responsive, whereas the R125Q variant is impaired and not AdoMet-responsive. Steady-state kinetics of the wild type and variant enzymes are consistent with a model that AdoMet activates CBS by inducing a conformational change of the enzyme that facilitates the substrates' access to the active site. Correlated changes in heme properties and catalytic chemistry in the variants are attributed to the disrupted hydrogen bonding and salt bridge interactions caused by the mutations. Analysis of the chemical mechanisms and structures of CBS and its closest homolog, cysteine synthase, suggested that two active-site tyrosine residues serve as proton donors that are critical for CBS activity, and may be used to distinguish CBS and cysteine synthase from their sequences. |
