Mono and dual cofactor dependence of human cystathionine β-synthase enzyme variants in vivo and in vitro.
Any two individuals differ from each other by an average of 3 million single-nucleotide polymorphisms. Some polymorphisms have a functional impact on cofactor-using enzymes and therefore represent points of possible therapeutic intervention through elevated-cofactor remediation. Because most known disease-causing mutations affect protein stability, we evaluated how the in vivo impact caused by single amino acid substitutions in a prototypical enzyme of this type compared with physical characteristics of the variant enzymes in vitro. We focused on cystathionine β-synthase (CBS) because of its clinical relevance in homocysteine metabolism and because some variants of the enzyme are clinically responsive to increased levels of its B6 cofactor. Single amino-acid substitutions throughout the CBS protein caused reduced function in vivo, and a subset of these altered sensitivity to limiting B6-cofactor. Some of these B6-sensitive substitutions also had altered sensitivity to limiting heme, another CBS cofactor. Limiting heme resulted in reduced incorporation of heme into these variants, and subsequently increased protease sensitivity of the enzyme in vitro. We hypothesize that these alleles caused a modest, yet significant, destabilization of the native state of the protein, and that the functional impact of the amino acid substitutions caused by these alleles can be influenced by cofactor(s) even when the affected amino acid is distant from the cofactor binding site.
The California Institute for Quantitative Biosciences, Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720.
Dago
D
Dimster-Denk
Katherine W
KW
Tripp
Nicholas J
NJ
Marini
Susan
S
Marqusee
Jasper
J
Rine
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003067:Coenzymes / Q000378:metabolism*; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D025101:Vitamin B 6 / Q000378:metabolism
D003067:Coenzymes; D006418:Heme; D025101:Vitamin B 6; D003541:Cystathionine beta-Synthase
CYS4; Saccharomyces; cystathionine β-synthase; cysteine biosynthesis; heme; vitamin B6