ABSTRACT
The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.
Subject(s)
Chitinases/metabolism , Euphorbiaceae/enzymology , Muramidase/metabolism , Acetylglucosamine/analogs & derivatives , Acetylglucosamine/chemistry , Acetylglucosamine/metabolism , Acetylglucosamine/pharmacology , Binding, Competitive , Chitinases/antagonists & inhibitors , Chromatography, High Pressure Liquid , Coloring Agents/metabolism , Kinetics , Muramidase/antagonists & inhibitors , Oligosaccharides/metabolism , Oxidation-Reduction , Plant Proteins , Thermodynamics , Trisaccharides/chemistry , Trisaccharides/pharmacologyABSTRACT
Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. In this paper the cleavage specificity of hevamine for peptidoglycan was studied by HPLC and mass-spectrometry analysis of enzymatic digests. The results clearly showed that the enzyme cleaves between the C-1 of a N-acetylglucosamine and the C-4 of a N-acetylmuramate residue. This means that hevamine, and very likely also other family 18 glycosyl hydrolases which cleave peptidoglycan, cannot be classified as lysozymes.
Subject(s)
Chitinases/metabolism , Muramidase/metabolism , Peptidoglycan/metabolism , Trees/enzymology , Acetylglucosamine/metabolism , Chromatography, High Pressure Liquid , Mass Spectrometry , Muramic Acids/chemistry , Muramic Acids/metabolism , Plant Proteins , Substrate SpecificityABSTRACT
Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced kcat and much higher Km values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.
Subject(s)
Bromides/metabolism , Chlorides/metabolism , Hydrolases/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Deuterium Oxide/pharmacology , Hydrolases/metabolism , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Structure-Activity Relationship , TryptophanABSTRACT
A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted Alu dimer repeat was identified further downstream.
