Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

• Published in: FEBS letters Vol. 478; no. 1; pp. 119 - 122
• Main Authors: Bokma, Evert, Barends, Thomas, Terwisscha van Scheltinga, Anke C., Dijkstra, Bauke W., Beintema, Jaap J.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 28.07.2000
• Subjects: Acetylglucosamine - analogs & derivatives; Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Acetylglucosamine - pharmacology; Binding, Competitive; Chitin oligomer; Chitinase; Chitinases - antagonists & inhibitors; Chitinases - metabolism; Chromatography, High Pressure Liquid; Coloring Agents - metabolism; Competitive inhibition; Euphorbiaceae - enzymology; Hevea brasiliensis; Kinetics; Muramidase - antagonists & inhibitors; Muramidase - metabolism; Oligosaccharides - metabolism; Oxidation-Reduction; Plant Proteins; Thermodynamics; Trisaccharides - chemistry; Trisaccharides - pharmacology; Chitinase; Hevea brasiliensis; Competitive inhibition; Chitin oligomer
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)01833-0

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 μM. Under the conditions tested, tetra-nag did not inhibit hevamine.