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

John G. Verburg, Shaukat H. Rangwala, Deborah A. Samac, Verne A. Luckow, Q. Khai Huynh

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Abstract

Using the catalytic mechanism of lysozyme as a paradigm for the mechanism of other enzymes that catalyze the hydrolysis of β-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.

Original languageEnglish (US)
Pages (from-to)223-230
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume300
Issue number1
DOIs
StatePublished - Jan 1 1993

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