Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin.
Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed chitinase A as the major species. Chitinase B' was a minor component of the chitinolytic complex. Whereas some properties were common to the three forms, analysis of other parameters revealed significant catalytic site-related differences. Accordingly, the three chitinases hydrolyzed the fluorogenic substrate 4-methylumbelliferyl chitotriose with typical Michaelian kinetics and Km values of 4.5, 11.8 and 3.8 microM for A, B and B', respectively. Allosamidin strongly inhibited the three enzyme forms with different kinetics. Dixon plots revealed competitive-type inhibition and Ki values of 10.0, 2.3 and 10.8 nM for A, B and B', respectively. Km/Ki ratios indicated 450-, 350- and 5130-fold higher affinity for the inhibitor over the substrate for the A, B and B' forms, respectively. Results are discussed in terms of the possibility that the three chitinase species correspond to different enzyme proteins.
Villagómez-Castro
J C
JC
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad Autónoma de Guanajuato, Gto, México.
López-Romero
E
E
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite / Q000378:metabolism; D004747:Entamoeba / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006027:Glycosides / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D014312:Trisaccharides / Q000378:metabolism / Q000494:pharmacology
C030561:4-methylumbelliferyl glycosides; D004791:Enzyme Inhibitors; D006027:Glycosides; D007527:Isoenzymes; D014312:Trisaccharides; C054851:allosamidin; D002686:Chitin; D006923:Hymecromone; C041161:chitotriose; D017886:Durapatite; D002688:Chitinases; D000117:Acetylglucosamine