Archives of Biochemistry and Biophysics 438 (2005) 146–155 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.04.019 Mechanistic analysis of wheat chlorophyllase Kiani A.J. Arkus a , Edgar B. Cahoon b,¤ , Joseph M. Jez a,¤ a Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO 63132, USA b United States Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, Donald Danforth Plant Science Center, 975 N. Warson Rd, St. Louis, MO 63132, USA Received 6 February 2005, and in revised form 25 April 2005 Available online 10 May 2005 Abstract Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripen- ing. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. PuriWcation 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 D 566 min ¡1 ; K m D 63 M) and was active over a broad temperature range (10–75 °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 »6.5 for both k cat and k cat /K m with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, sol- vent kinetic isotope eVects 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 inven- tory 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 mecha- nism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.  2005 Elsevier Inc. All rights reserved. Keywords: Chlorophyll; Chlorophyllase; Triticum aestivum; Plant senescence; Hydrolase; Reaction mechanism Chlorophyll is the most abundant pigment on Earth with an estimated one billion tons degraded each year [1]. Chlorophyll breakdown occurs during the normal turnover of the pigment, when leaves change color in autumn [2], at speciWc developmental stages, including leaf senescence and fruit ripening [3–7], and in cell death triggered by environmental factors such as extreme tem- peratures or water shortage [8,9]. Degradation of the pigment is highly regulated to prevent cellular damage by the photodynamic action of chlorophyll and any of its colored derivatives [1,2]. A series of enzyme-catalyzed reactions transform chlorophyll into its colorless Wnal form [9]. Chlorophyllase (chlorophyll-chlorophyllidohy- drolase, EC 3.1.1.14) catalyzes the Wrst step in this pro- cess, i.e., the hydrolysis of chlorophyll to chlorophyllide and phytol (Fig. 1). Over 90 years ago, chlorophyllase was one of the Wrst plant enzymes identiWed biochemi- cally [10], and its enzymatic activity is widespread in plant and algal species [1,2,9,11]. Recently, a role for chlorophyllase in protecting plant tissues from oxidative damage and in modulating the activation of diVerent plant defense pathways has been proposed [12]. The biological function of chlorophyllase has poten- tial industrial and agricultural applicability. Slowing the rate of chlorophyll degradation either by gene manipula- tion or small molecule inhibitors would maintain the green color and fresh appearance of plants or plant material and may improve cereal crop yields by delaying * Corresponding authors. E-mail addresses: ecahoon@danforthcenter.org (E.B. Cahoon), jjez@danforthcenter.org (J.M. Jez).