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 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.
Subject(s)
Chitinases/metabolism , Coccidioides/enzymology , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Acetylglucosamine/metabolism , Binding Sites , Coccidioides/pathogenicity , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Oligosaccharides/metabolism , Substrate Specificity , Time FactorsABSTRACT
The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.
Subject(s)
Chitinases/chemistry , Coccidioides/chemistry , Acetylglucosamine/analogs & derivatives , Acetylglucosamine/chemistry , Amino Acid Sequence , Binding Sites , Chitinases/antagonists & inhibitors , Chitinases/genetics , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Trisaccharides/chemistryABSTRACT
Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state.
Subject(s)
Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Protein Structure, Tertiary , Codon, Terminator , Cytochrome c Group/metabolism , Enzyme Stability , Leucine , Mutation, Missense , Phenotype , Protein Folding , Yeasts/geneticsABSTRACT
Chitinase is necessary for fungal growth and cell division and, therefore, is an ideal target for the design of inhibitors which may act as antifungal agents. A chitinase from the fungal pathogen Coccidioides immitis has been expressed as a fusion protein with gluathione-S-transferase (GST), which aids in purification. After cleavage from GST, chitinase was crystallized from 30% PEG 4000 in 0. 1 M sodium acetate pH 4.6. The crystals have a tetragonal crystal lattice and belong to space group P41212 or P43212 and diffract to 2. 2 A resolution. The unit-cell parameters are a = b = 91.2, c = 95.4 A; there is only one chitinase molecule in the asymmetric unit.