Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human cystathionine beta-synthase.
Human cystathionine beta-synthase is a heme protein that catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate-dependent reaction. Mutations in this enzyme are the leading cause of hereditary hyperhomocysteinemia with attendant cardiovascular and other complications. The enzyme is activated approximately 2-fold by the allosteric regulator S-adenosylmethionine (AdoMet), which is presumed to bind to the C-terminal regulatory domain. The regulatory domain exerts an inhibitory effect on the enzyme, and its deletion is correlated with a 2-fold increase in catalytic activity and loss of responsiveness to AdoMet. A mutation in the C-terminal regulatory domain, D444N, displays high levels of enzyme activity, yet is pathogenic. In this study, we have characterized the biochemical penalties associated with this mutation and demonstrate that it is associated with a 4-fold lower steady-state level of cystathionine beta-synthase in a fibroblast cell line that is homozygous for the D444N mutation. The activity of the recombinant D444N enzyme mimics the activity of the wild-type enzyme seen in the presence of AdoMet and can be further activated approximately 2-fold in the presence of supraphysiolgical concentrations of the allosteric regulator. The mutation increases the K(act) for AdoMet from 7.4 +/- 0.2 to 460 +/- 130 microM, thus rendering the enzyme functionally unresponsive to AdoMet under physiological concentrations. These results indicate that the D444N mutation partially abrogates the intrasteric inhibition imposed by the C-terminal domain. We propose a model that takes into account the three kinetically distinguishable states that are observed with human cystathionine beta-synthase: "basal" (i.e., wild-type enzyme as isolated), "activated" (wild-type enzyme + AdoMet or the D444N mutant as isolated), and superactivated (D444N mutant + AdoMet or wild-type enzyme lacking the C-terminal regulatory domain).
Evande
Ruby
R
Biochemistry Department, University of Nebraska, Lincoln, NE 68588-0664, USA.
Blom
Henk
H
Boers
Godfried H J
GH
Banerjee
Ruma
R
D000494:Allosteric Regulation / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry; D002460:Cell Line; D003541:Cystathionine beta-Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005296:Ferrous Compounds / Q000737:chemistry; D005347:Fibroblasts / Q000201:enzymology; D006418:Heme / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000494:pharmacology
D005296:Ferrous Compounds; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006418:Heme; D001216:Asparagine; D012436:S-Adenosylmethionine; D002248:Carbon Monoxide; D003541:Cystathionine beta-Synthase