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Heparin Lyase EC.4.2.2.7

Heparin Lyase EC.4.2.2.7. Hendrikus MBB 20510002. Enzyme Nomenclature. PARAMETER FUNGSIONAL (all Organism). STRUKTUR HEPARIN. Composed of primarily of 2-O-sulfo-a-L-iduronic acid and 2-deoxy-2-sulfamino-6-O-sulfo-a-D-glucose. Heparin Lyase.

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Heparin Lyase EC.4.2.2.7

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  1. Heparin LyaseEC.4.2.2.7 Hendrikus MBB 20510002

  2. Enzyme Nomenclature

  3. PARAMETER FUNGSIONAL (all Organism)

  4. STRUKTUR HEPARIN • Composed of primarily of 2-O-sulfo-a-L-iduronic acid and 2-deoxy-2-sulfamino-6-O-sulfo-a-D-glucose

  5. Heparin Lyase • HL membantu pemahaman mengenai struktur heterogen yang berhubungan dengan peran fisiologis heparin dan heparan sulfat sebagai antikoagulasi, potensiasi angiogenesis, modulasi proliferasi seluler dan aplikasi klinis sebagai monitoring level heparin dan neutralisasi heparin dalam darah.

  6. STRUKTUR HEPARINASE I Schematic representation of the heparinase I-H151A-heparin oligosaccharide (HE12) complex. The heparin is shown in stick representation, and Ca2' is shown as an orange sphere. The thumb domain is colored blue, and the tip of the thumb (indicated by the blue arrow) is colored cyan. Heparinase I terdiri atas domain dengan gambaran “jellyroll” yang terbentuk dari 2 bentuk concave "-sheets (eight antiparallel "-strands each) yang memeiliki lipatan glukanase dan domain thumb-like insertion dari salah satu sisi.

  7. STRUKTUR HEPARINASE I Close-up of the thumb domain rainbow (blue, N terminus; red, C terminus). Domain dengan gambaran “thumb” memiliki bentuk yang tidak biasa dari bentukan 5 bagian pendek antiparalel. Gambaran menyerupai susunan segitiga dengan dua rantai panjang dan 1 rantai pendek.

  8. STRUKTUR HEPARINASE I Bagian dalam “jellyroll” melekuk sempurna, membentuk seperti jurang yang memanjang melintasi rantainya. Domain “thumb” bermuatan positif, terutama pada sisi “jurang” nya dan sekaligus sebagai tempat berikatan dengan muatan negatig heparin.

  9. STRUKTUR HEPARINASE I • Bagian yang curam secara partial ditutup oleh gambaran seperti kelopak yang dibentuk oleh 2 loop, * Gly74-Glu79 and Thr250-Asn257 dari domain “jellyroll” * Lys184-Val187 dari domain “thumb” • Integritas struktur heparinase I dipertahankan oleh ion Ca2+ yang terletak antara domain “jellyroll” dan domain “thumb” dan berhubungan dengan rantai samping dari beberpa loop.

  10. STRUKTUR HEPARINASE I • Ligan equatorial dari bipyramid pentagonal yaitu - O*1 (2.8 Å) dan O*2 (2.6 Å) dari Glu222, - O*2 (2.3 Å) dari Asp346, - dan backbone carbonyl oxygens dari Trp248 (2.4 Å) dan Asn345 (2.5 Å). • Aksis ligand bipyramid yaitu: - O*1 dari Asp155 (2.8 Å) and - satu molekul air (2.6 Å) Bipyramidal coordination of a Ca2' ion in apo heparinase I structure. These and subsequent figures were prepared with PyMOL

  11. MEKANISME REAKSI Degradation of heparin and heparin sulfate by heparinases I–III. The arrows indicate the cleavage site.

  12. Heparin Lyase (3IN9)Bacteroides thetaiotaomicron ISOLASI: Flavobacterium heparinum , Bacteroides thetaiotaomicron dan Baceroides stercoris. Gambar Struktur kristal heparin lyase dari Bacteroides thetaiotaomicron.

  13. ISOLASI & PEMURNIAN Dicuci dng 500 ml (0.025M)Tris,pH 7.9 in 0.1M Nacl 6.8 gr extract Flavobacteria Supernatan Gradient Penambahan Protamine Sulfat (10mg/ml Lar); Dicampur pelan + 100ml (0.5M)NaAcetat pH 6.4 Penambahan 500ml (0.025 M) Tris dalam 0.7 M NaCl pH7.9 Sentrifugasi 25.000g;30 mnt Masukan dlm Vessel yg mengandung 500ml (0,025M)Tris in 0,1 M NaCl, pH 7.9 Supernatan (diencerkan dng 0.05M asetat dan di + kan 35gr Phosposelulosa pd Kolumn (30x2.1 cm) Fraksi 6ml (aktivitas Heparinase) Sentrifugasi 25.000 g; 30 menit Ultrafiltrasi Supernatan Enzim

  14. ISOLASI DAN PURIFIKASI • Temperatur Optimum : 31oC • pH optimum : 6.5 • Penyimpanan : Stabil selama 1 bulan pada -15oC pada 60µg Protein per mL buffer. Pada 4oC bertahan selama 5 minggu.

  15. STIMULASI DAN INHIBISI Stimulation (slight) : Ca++, Mn++, Cd++ sedangkan Mg++, Ba++ dan Zn++ tidak berefek pada 10-3 M Inhibitor :Hg++ (100%), Cu++ dan Fe+++ (50% pada 10-5 M)

  16. APLIKASI Heparin is generally used for anticoagulation for the following conditions: Acute coronary syndrome, e.g., NSTEMI Atrial fibrillation Deep-vein thrombosis and pulmonary embolism Cardiopulmonary bypass for heart surgery. ECMO circuit for extracorporeal life support Haemofiltration

  17. Aplikasi

  18. Biological Activities and Therapeutic Potential Melalui pengaturan aktivitas heparin-binding proteins, heparin dapat mempengaruhi berbagai proses biologis sehingga memiliki aplikasi terapeutik sebagai : antithrombotik, antiatherosclerotic, anticomplement, antiinfective, anticancer, and Antiinflammatory agent. The heparin-binding proteins berperan sebagai target terapeutik baik sebagai enzymes, protease inhibitors, lipoproteins, growth factors, chemokines, selectins, extracellular matrix proteins, receptor proteins, viral coat proteins, dan nuclear proteins

  19. Biological Activities and Therapeutic Potential

  20. EFEK BIOMEDIS

  21. EFEK BIOMEDIS(2)

  22. EFEK BIOMEDIS(3)

  23. EFEK BIOMEDIS(4)

  24. EFEK BIOMEDIS(5)

  25. APLIKASI BIOTEKNOLOGI Dalam bidang industri HL dapat diaplikasikan untuk membuat heparin dengan berat molekul rendah sehingga dapat disintesis untuk obat-obatan. Bacteroides thetaiotaomicron : enzyme dengan kemurnian tinggi dan aktivitas spesifik dapat bermanfaat dalam pengembangan obat yang digunakan scara klinis. Flavobacterium heparinum : Pengembangan dengan metode kolorimetrik yang mampu mendeteksi aktivitas heparinase sehingga dapat diidentifikasi sebagai obat anti metastasi masa depan dan obat anti imflamasi.

  26. Referrence • Han, Young-Hyun, et.al. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 49, pp. 34019–34027, December 4, 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. • Hovigngh, P and A.Linker. The Enzymatic Degradation of Heparin and Heparitin Sulfate. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 22, Issue of November 25, pp. 6170-6175, 1970 . Printed in U.S.A. • Kim, Wan-Seok, et.al. Purification and Characterization of Heparin Lyase I from Bacteroides stercoris HJ-15. Journal of Biochemistry and Molecular Biology, Vol. 37, No. 6, November 2004, pp. 684-690. • Munoz, Eva M and Robert J.L. Heparin Binding Domains in Vascular Biology. Arterioscler Thromb Vasc Biol 2004;24;1549-1557; originally published online Jul 1,2004 by American Heart Association. • Xie, Jin.et.al. Physiological, Pathophysiological and Therapeutic Roles of Heparin and Heparan Sulfate. In Carbohydrate Chemistry, Biology and Medical Applications • Hari G. Garg, Mary K. Cowman and Charles A. Hales. Page 229 – 253. • Murugesan S and Robert J.L. Immobilization of Heparin: Approaches and Applications. Current Topics in Medicinal Chemistry, 2008, 8, 80-100.

  27. Referrence • Powell, Andrew K.et.al. Interactions of heparin/heparan sulfate with proteins: Appraisal of structural factors and experimental approaches. Glycobiology vol. 14 no. 4 pp. 17-30, 2004 • Shaya, David.et.al. Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 26, pp. 20051–20061, June 25, 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. • Luo Y, Huang X, McKeehan WLHigh yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli. Arch Biochem Biophys. 2007 Apr 1;460(1):17-24. Epub 2007 Feb 16. • Ahn SC, Kim BY, Oh WK, Park YM, Kim HM, Ahn JS. Colorimetric heparinase assay for alternative anti-metastatic activity. Life Sci. 2006 Sep 20;79(17):1661-5. Epub 2006 May 27. • EC 4.2.2.7 Heparin Lyase. Diunduh dari http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.2.7 • IUBMB Enzyme Nomenclature: EC 4.2.2.7. Diunduh dari http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/2/2/7.html

  28. Terima Kasih

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