Examination of the role of tyrosine-174 in the catalytic mechanism of the Arabidopsis thaliana chitinase: comparison of variant chitinases generated by site-directed mutagenesis and expressed in insect cells using baculovirus vectors.
Using the catalytic mechanism of lysozyme as a paradigm for the mechanism of other enzymes that catalyze the hydrolysis of beta-1,4-glycosidic linkages, including chitinase, we have examined the effect of chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) on the reaction catalyzed by Zea mays chitinase. Inactivation with EDC did not result in derivatization of essential carboxylic acid residues, but resulted in the selective modification of a single essential tyrosine residue (Verburg, J. G., Smith, C. E., Lisek, C. A., and Huynh, Q. K., 1991, J. Biol. Chem. 267, 3886-3893). Here, we examine the role of the homologous tyrosine residue in the catalytic mechanism of the Arabidopsis thaliana chitinase. Tyrosine-174 of the Arabidopsis chitinase was replaced, with phenylalanine, alanine, histidine, and methionine by site-directed mutagenesis, and the variant chitinases were expressed in insect cells using baculovirus transfer vectors. A comparison of the reaction catalyzed by each of the variant enzymes indicates that substitution of another amino acid for Tyr-174 alters, but does not eliminate, enzymatic activity. Estimates of the specific activities of the variant chitinases reveal that substitution of His for Tyr-174 has a minimal effect on catalysis, the specific activities of the Phe and Met variants are approximately equivalent to each other, but are 60% the specific activity of wild-type Arabidopsis chitinase, and the specific activity of the Ala variant is only 40% that of wild-type. The observation that the Arabidopsis chitinase is tolerant to mutagenesis at this position suggests that Tyr-174 does not participate directly in catalysis.
Verburg
J G
JG
Department of Protein Biochemistry, Monsanto Agricultural Company, Chesterfield, Missouri 63198.
Rangwala
S H
SH
Samac
D A
DA
Luckow
V A
VA
Huynh
Q K
QK
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014644:Genetic Variation; D005822:Genetic Vectors; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D002688:Chitinases