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 cata lytic mechanism; that is, the beta -conformation of the chitin substrate li nkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglu cosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc)( 6) is predominantly cleaved into (GlcNAc)(2) and (GlcNAc)(4), where the (Gl cNAc)(2) group arises from the nonreducing end of the substrate and is form ed as the beta -anomer with time, transglycosylation occurs, generating (Gl cNAc)(8) from the product dimer and fresh hexamer. Similar patterns are see n 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 muM. Synthetic substrates with fluorescent leaving group s exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of k inetic constants. An improved theoretical analysis of the time-course of (G lcNAc)(6) degradation allows the unitary free energy of binding of the indi vidual subsites of the enzyme to be estimated. The free energy values obtai ned are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tes ted against the crystal structure of the enzyme.