Kinetic Properties of Chitinase-1 from the Fungal Pathogen Coccidioides immitis

Abstract

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 -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 -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 µM. 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.

Determination of substrate binding energies in individual subsites of a family 18 chitinase

“…Apparently, these interactions are not only necessary to overcome the loss of free energy associated with the distortion of the sugar bound in the À1 subsite from the 4 C 1 chair conformation to the twisted 1,4 B boat conformation (calculated to amount to $8 kcal/mol [27]), but also to drive the actual binding. Based on theoretical analysis of kinetic data for substrate conversion, a free energy change of +3.1 kcal/mol has been calculated for the À1 subsite in chitinase-1 from the fungal pathogen Coccidioides immitis [12]. A positive DG r for sugar binding in the À1 subsite is to be expected due to the loss of free energy upon sugar distortion, and is in agreement with observations made in classical studies of substrate affinities in lysozyme [28].…”

“…Substratebinding studies in family 18 chitinases [12][13][14] and family 19 chitinases [15] have been described previously, but several of these were based on modelling only. Very recently, direct measurements of binding affinities have been described for Chit-42 from Trichoderma harzianum and for ChiB from Serratia marcescens [13,14].…”

Mutation of a Conserved Tryptophan in the Chitin-Binding Cleft ofSerratia marcescensChitinase A Enhances Transglycosylation

“…In the active site, this tetrasaccharide would then bind from À2 to +2 and become rapidly hydrolyzed to two molecules of disaccharide. Fukamizo et al 15) calculated for a homologous family 18 chitinase from Coccidioides immitis that binding at À2 is favored by about À3:8 kcal/mole, which would promote the shift of the newly formed (GlcNAc)4 across À2 to +2 subsites.…”

“…This probably occurs because a very weak trisaccharide substrate is transformed by transglycosylation into a very reactive tetrasaccharide, which has high affinity for binding productively from À2 to +2. 15) During hydrolysis of (GlcNAc)4 by SmChiA W167A , the intermediate positive glycosyl fragment (GlcNAc)2 + that is formed might also be retained long enough to serve as a donor during transglycosylation. This reaction behavior is apparently promoted when (GlcNAc)4 is incubated with an inactive pseudotrisaccharide acceptor pNp-(GlcNAc)2.…”

Role of Tryptophan Residues in a Class V Chitinase fromNicotiana tabacum

“…The reaction products from the chitinase-catalyzed hydrolysis of (GlcNAc) n (n ¼ 4, 5, or 6) were determined quantitatively by gel filtration HPLC by the method of Fukamizo et al 19) The enzymatic reaction was performed in 20 mM sodium acetate buffer, pH 5.0, at 40 C. The enzyme and substrate concentrations were 0.32 mM and 4.6 mM, respectively. To terminate the enzymatic reaction completely at a given incubation time, a portion of the reaction mixture was mixed with an equal volume of 0.1 M NaOH solution, and immediately frozen in liquid nitrogen.…”