Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin.
Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.
Milewski
S
S
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Poland.
O'Donnell
R W
RW
Gooday
G W
GW
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001224:Aspartic Acid; D001665:Binding Sites; D001667:Binding, Competitive; D002176:Candida albicans / Q000201:enzymology*; D002688:Chitinases / Q000037:antagonists & inhibitors*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D010658:Phenylglyoxal / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014312:Trisaccharides / Q000494:pharmacology*
D005971:Glutamates; D013439:Sulfhydryl Reagents; D014312:Trisaccharides; C054851:allosamidin; D001224:Aspartic Acid; D018698:Glutamic Acid; D002688:Chitinases; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D005022:Ethyldimethylaminopropyl Carbodiimide; D000117:Acetylglucosamine