Abstract
The first committed step of transsulfuration is catalyzed by cystathionine beta-synthase (CBS), a known pyridoxal 5'-phosphate (PLP) enzyme. The inferred amino acid sequences of rat liver CBS and rat liver hemoprotein H-450 are identical. We now confirm the presence of heme b in rat and human liver CBS. Heme almost entirely accounts for the visible spectrum of CBS rather than PLP. Human CBS, expressed in Escherichia coli, acquires heme b from the host bacteria. delta-Aminolevulinate supplementation during bacterial growth increases both the heme saturation and the specific activity of the homogeneous enzyme more than 3-fold. 1 mol of the 63-kDa CBS subunit binds 1 mol of each (heme and PLP). The presence of heme is required for PLP binding, and the amount of PLP bound is limited by the heme content. Removal of PLP, but not heme, from CBS is reversible. These findings suggest that heme is functionally incorporated into CBS only during protein folding. This report describes the first instance of an enzyme that depends upon both heme and PLP for its function.
Highlights
The first committed step of transsulfuration is cata- tural change is accompanied by a substantial increase in enlyzed by cystathionine P-synthase (CBS), kanown pyri- zyme specific activity, due almost entirely toa decrease in the doxal 5”phosphate (PLP) enzyme
Acid sequences of rat liver CBS and rat liver hemopro- We purified the dimeric form of human liver CBS to homotein H-450are identical.We confirm the presencoef geneity and characterized it (Kraus and Rosenberg, 1983)
Rat CBS Is a Heme Protein-To investigate the possibility that CBS is a heme protein, we compared the spectral characteristics of immunoprecipitated rat liver CBS with the published characteristics of “hemoprotein H-450” (Kim and Deal, 1976; Kim, 1982; Omura et al, 1984)and other heme proteins (Berry and Trumpower, 1987)
Summary
The first committed step of transsulfuration is cata- tural change is accompanied by a substantial increase in enlyzed by cystathionine P-synthase (CBS), kanown pyri- zyme specific activity, due almost entirely toa decrease in the doxal 5”phosphate (PLP) enzyme. CBS and activates 2-4-fold the rat (Roper and Kraus, 1992) and human (Kozich and Kraus, 1992; Bukovska et al, 1994) enzymes. These data indicate that thenative rat CBS is a heme protein and that thheeme moiety is heme b, whichis present in the isolated native enzyme in its oxidized form.
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