Identification of an essential tyrosine residue in the catalytic site of a chitinase isolated from Zea mays that is selectively modified during inactivation with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

Abstract

Chitinase isolated from Zea mays seeds is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the absence of exogenous nucleophiles. Oli-gomers of N-acetylglucosamine, N,N′ ,N″,N‴-tetra-N-acetylchitotetraose (GIcNAc4), and to a lesser extent, N,N′,N″-tri-N-acetylchitotriose (GlcNAc3) and N,N′-di-N-acetylchitobiose (GlcNAc2) provide partial protection against inactivation by the reagent. An examination of the concentration dependence of the protection afforded by GIcNAc4 revealed direct competition between the substrate analog and the reagent for the same binding sites on the enzyme. Isolation and Kdman degradation of a "new" tryptic fragment, observed after inactivation of chitinase with EDC, revealed the sequence G-P-L-Q-I-S-W-N-*-N-Y-G-P-A-G-R, where the asterisk represents a cycle in which no amino acid was detected, presumably as a consequence of derivatization with EDC. In basic chitinases from dicotyledonous plants such as Arabidopsis thaliana, Phaseolis vulgaris (bean), Nicotiana tabacum (tobacco), and Solanum tuberosum (potato), as well as in the chitinase isolated from the monocotyledonous plant Hordeum vulgare (barley), this position is invariably occupied by a tyrosine. However, in the Oryza saliva (rice) basic chitinase, this position is occupied by a phenylalanine. The following additional evidence supports identification of this residue as tyrosine in Z. mays chitinase. (a) Inactivation of chitinase with EDC is reversible by treatment with hydroxylamine. (6) Liquid secondary ion mass spectrometric analysis of the isolated derivatized peptide revealed the presence of a molecular ion with a mass to charge ratio consistent with the peptide containing a derivatized tyrosine residue. These results provide evidence for an essential tyrosine residue at or near the catalytic site of chitinase that is selectively modified during inactivation with EDC.