On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+.
The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca(2+)-heparin, in which α-L-iduronate-2-O-sulfate residues adopt (1)C4 conformation preferentially, is a substrate, while Na(+)-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates β-elimination by abstracting the C5 proton of the α-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.
Disciplina de Biologia Molecular, Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio, 100, CEP 04044-020, São Paulo, SP, Brazil. hbnader.bioq@epm.br ivarne@umc.br.
Carolina R
CR
Córdula
Marcelo A
MA
Lima
Samuel K
SK
Shinjo
Tarsis F
TF
Gesteira
Laércio
L
Pol-Fachin
Vivien J
VJ
Coulson-Thomas
Hugo
H
Verli
Edwin A
EA
Yates
Timothy R
TR
Rudd
Maria A S
MA
Pinhal
Leny
L
Toma
Carl P
CP
Dietrich
Helena B
HB
Nader
Ivarne L S
IL
Tersariol
D001439:Bacteroides / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D006493:Heparin / Q000737:chemistry; D019763:Heparin Lyase / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011509:Proteoglycans; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D011509:Proteoglycans; D014443:Tyrosine; D006639:Histidine; D006493:Heparin; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D002118:Calcium