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- Title
Substrate specificity of Chondroitinase ABC I based on analyses of biochemical reactions and crystal structures in complex with disaccharides.
- Authors
Takashima, Makoto; Watanabe, Ippei; Miyanaga, Akimasa; Eguchi, Tadashi
- Abstract
Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the β-1,4 glycosidic linkage of chondroitin sulfate (CS) by β-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g. approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4- O -sulfated N -acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6- O -sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the k cat/ K m of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4- O -sulfated and 6- O -sulfated GalNAc residues. The distance between Arg500 and the 4- O -sulfate group was 0.8 Å shorter than that between Arg500 and the 6- O -sulfated group. Moreover, it is likely that the 6- O -sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4- O -sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I.
- Subjects
DISACCHARIDES; CRYSTAL structure; CHONDROITINASE; CHONDROITIN sulfates; CHONDROITIN sulfate proteoglycan; GALACTOSAMINE; ISOMERS
- Publication
Glycobiology, 2021, Vol 31, Issue 11, p1571
- ISSN
0959-6658
- Publication type
Article
- DOI
10.1093/glycob/cwab086