Purification and characterization of cystathionine β-synthase bearing a cobalt protoporphyrin.
Human cystathionine β-synthase (CBS), a pivotal enzyme in the metabolism of homocysteine, is a pyridoxal-5'-phosphate-dependent enzyme that also contains heme, a second cofactor whose function is still unclear. One strategy for elucidation of heme function is its replacement with different metalloporphyrins or with porphyrins containing different substituent groups. This paper describes a novel expression approach and purification of cobalt CBS (CoCBS), which results in a high yield of fully active, high purity enzyme, in which heme is substituted by Co-protoporphyrin IX (CoPPIX). Metal content analysis showed that the enzyme contained 92% cobalt and 8% iron. CoCBS was indistinguishable from wild-type FeCBS in its activity, tetrameric oligomerization, PLP saturation and responsiveness to the allosteric activator, S-adenosyl-l-methionine. The observed biochemical and spectral characteristics of CoCBS provide further support for the suggestion that heme is involved in structural integrity and folding of this unusual enzyme.
Department of Pediatrics and the Colorado Intellectual and Developmental Disabilities Research Center (IDDRC), University of Colorado at Denver, 12800 E 19th Ave., Aurora, CO 80045, USA.
Tomas
T
Majtan
Katherine M
KM
Freeman
Aaron T
AT
Smith
Judith N
JN
Burstyn
Jan P
JP
Kraus
D005591:Chemical Fractionation / Q000379:methods*; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D011524:Protoporphyrins; D013057:Spectrum Analysis
D011524:Protoporphyrins; C007095:cobaltiprotoporphyrin; D003541:Cystathionine beta-Synthase
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