Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis.
The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.
Bokma
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Department of Biochemistry, Rijksuniversiteit Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. e.bokma@chem.rug.nl
Barends
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Terwissch van Scheltingab
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Dijkstr
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Beintema
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D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000378:metabolism; D019665:Euphorbiaceae / Q000201:enzymology*; D007700:Kinetics; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins; D013816:Thermodynamics; D014312:Trisaccharides / Q000737:chemistry / Q000494:pharmacology
D004396:Coloring Agents; D009844:Oligosaccharides; D010940:Plant Proteins; D014312:Trisaccharides; C054851:allosamidin; C012238:chitotetrose; D002688:Chitinases; C063764:hevamine; D009113:Muramidase; D000117:Acetylglucosamine