Mechanistic analysis of wheat chlorophyllase.
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 a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, 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 pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton 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.
Arkus
Kiani A J
KA
Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
Cahoon
Edgar B
EB
Jez
Joseph M
JM
D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002734:Chlorophyll / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011522:Protons; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014908:Triticum / Q000201:enzymology*
D011522:Protons; D012997:Solvents; D002734:Chlorophyll; D006867:Hydrolases; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase