Heparinases: clonagem, expressão e requisitos estruturais para a atividade

• Ano de defesa: 2011
• Autor(a) principal: Córdula, Carolina Ribeiro [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: clonagem; enzima; heparina; heparinase
• Link de acesso: http://repositorio.unifesp.br/handle/11600/9688

Structural characteristics of heparin (Hep) and heparan sulfate (HS) have been determined using enzymes from Flavobacterium heparinum, a non-pathogenic soil bacterium. Upon induction with Hep/HS or their disaccharides as the sole source of carbon and nitrogen, the bacteria synthesize heparinase, and heparitinases I and II. This lyases cleaves heparin and HS by a -eliminative process in a random endolytic pattern. Heparitinase I cleaves exclusively Nacetyl or N-sulfo-glucosaminide-glucuronic acid linkage of HS/Hep without C6 sulfate substitution; Heparitinase II cleaves N-acetyl-6-sulfo or N-sulfo or N,6- sulfo glucosaminide-glucuronic/ iduronic acid linkage of HS/Hep without C3 sulfate substitution. Heparinase cleaves an -D-glucosamine N- and 6-sulfated (14) -L-iduronate 2-sulfated, present in heparin. This work aims to cloning and express heparinase and heparitinase I and study de kinetic behavior of heparinase. Recombinant enzymes were expressed in E. coli using the T7 polymerase pET expression system. The work shows that heparin-Ca2+- heparinase I complex is the true substrate for heparinase (KS = 1.4±0.1μM), whereas heparin Ca2+ free (KI=12±2μM) as an important pharmaceutical contaminant of commercial heparin, oversulfate chondroitin sulfate (OSCS) (Ki= 0.65μM) are competitive inhibitors. The pKa values of the prototropic groups of the active site were determined by measuring the pH-, solvent- and temperature-dependence upon kcat/KSA, kcat and KSA constants. The results show, at pHoptimium=6.67±0.05, a deprotonated histidine residue initiating the - elimination reaction by the abstraction of the C5 proton of the -L-iduronate 2- O-sulfate residue; and a tyrosin residue in a protonated form acting as a proton donor to the hexosamine leaving group. Mutations of histidine 165 and glutamine 163 residue were performed and kinetic analysis show specific activity maintenance (5,0 e-007 Abs/μg.s), suggesting that H165 and Q163 were not essencial for the heparinase activity.