Kinetic properties of chitinase-1 from the fungal pathogen Coccidioides immitis.
The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the beta-conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc)6 is predominantly cleaved into (GlcNAc)2 and (GlcNAc)4, where the (GlcNAc)2 group arises from the nonreducing end of the substrate and is formed as the beta-anomer. With time, transglycosylation occurs, generating (GlcNAc)8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc)5 and (GlcNAc)4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc)4 is 50 microM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc)6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.
Fukamizo
T
T
Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Sasaki
C
C
Schelp
E
E
Bortone
K
K
Robertus
J D
JD
D000117:Acetylglucosamine / Q000378:metabolism; D001665:Binding Sites; D002688:Chitinases / Q000378:metabolism*; D003045:Coccidioides / Q000201:enzymology* / Q000472:pathogenicity; D005656:Fungal Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013997:Time Factors
D005656:Fungal Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C121990:CTS1 protein, S cerevisiae; D002688:Chitinases; D000117:Acetylglucosamine