Reference "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"

Reference ID

4758

Title

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

Source

The Journal of biological chemistry, 1992, vol. 267, pp. 3886-3893

Authors (4)

Verburg-J-G	Smith-C-E
Lisek-C-A	Huynh-Q-K

Abstract

Chitinase isolated from Zea mays seeds is inactivated by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) in the absence of exogenous nucleophiles.
Oligomers of N-acetylglucosamine,N,N',N",N"'-tetra-N- acetylchitotetraose
(GlcNAc4), 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 GlcNAc4 revealed direct competition between the
substrate analog and the reagent for the same binding sites on the enzyme.
Isolation and Edman 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 sativa
(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. (b) 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.