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Mechanistic Analysis of Wheat Chlorophyllase
Author(s) -
Arkus Kiani Anela Jeniah,
Cahoon Edgar,
Jez Joseph
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a40-a
Subject(s) - enzyme kinetics , chemistry , enzyme , chlorophyll , enzyme assay , active site , hydrolysis , stereochemistry , biochemistry , organic chemistry
Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide the first detailed mechanistic analysis of the enzyme from any source. Purification of recombinant chlorophyllase from an E. coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (kcat=566 min‐1; Km=63 ?M) and was active over a broad temperature range (10–75 °C). For the first time, we demonstrate that the enzyme displays carboxylesterase activity toward p‐nitrophenyl (PNP)‐butyrate, PNP‐decanoate, and PNP‐palmitate. The pH‐dependence of the reaction showed the involvement of an active site residue with a pKa of ~6.5 for both kcat and kcat/Km with chlorophyll, PNP‐butyrate, and PNP‐decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5–1.9 and 1.4–1.9 on kcat and kcat/Km, respectively, were observed. Proton inventory experiments suggest that a single proton is transferred in the rate‐limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge‐relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.