Coenzyme B12 dependent diol dehydratase undergoes mechanism‐based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [Biochemistry16, 1082–1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro‐S‐CH2OH group is oriented to the hydrogen‐abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X‐ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)‐1,2‐propanediol, i.e. in the GS conformation, with its 3‐OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild‐type enzyme. kcat/kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild‐type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)‐1,2‐propanediol decreased on these mutations. The substrate activities towards longer chain 1,2‐diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2‐diols.
Database
Structural data are available in the Protein Data Bank under the accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3AUJ.
Structured digital abstract
http://www.uniprot.org/uniprot/Q59472, http://www.uniprot.org/uniprot/Q59471 and http://www.uniprot.org/uniprot/Q59470 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8301985)
Systemic mastocytosis is a disease of mast cell proliferation that may be associated with hematologic disorders. There are no features on examination that allow the diagnosis of systemic disease, and mast cell–derived mediators, which may be elevated in urine or blood, may also be elevated in individuals with severe allergic disorders. Thus, the diagnosis usually depends on results of bone marrow biopsy. To facilitate evaluation, surrogate markers of the extent and severity of the disease are needed. Because of the association of mastocytosis with hematologic disease, plasma levels were measured for soluble KIT (sKIT) and soluble interleukin-2 receptor alpha chain (sCD25), which are known to be cleaved in part from the mast cell surface and are elevated in some hematologic malignancies. Results revealed that levels of both soluble receptors are increased in systemic mastocytosis. Median plasma sKIT concentrations as expressed by AU/mL (1 AU = 1.4 ng/mL) were as follows: controls, 176 (n = 60); urticaria pigmentosa without systemic involvement, 194 (n = 8); systemic indolent mastocytosis, 511 (n = 30); systemic mastocytosis with an associated hematologic disorder, 1320 (n = 7); aggressive mastocytosis, 3390 (n = 3). Plasma sCD25 levels were elevated in systemic mastocytosis; the highest levels were associated with extensive bone marrow involvement. Levels of sKIT correlated with total tryptase levels, sCD25 levels, and bone marrow pathology. These results demonstrate that sKIT and sCD25 are useful surrogate markers of disease severity in patients with mastocytosis and should aid in diagnosis, in the selection of those needing a bone marrow biopsy, and in the documentation of disease progression.
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