Enzymatic conversion of pheophorbide a to the precursor of pyropheophorbide a in leaves of Chenopodium album

Abstract

Soluble proteins extracted from leaves of Chenopodium album catalyzed the conversion of pheophorbide a to a precursor of pyropheopborbide a, putatively identified as C-132-carboxyl-pyropheophorbide a. The precursor was then decarbuxylated non-enzymatically to yield pyropheophorbide a. Soluble proteins and pheophorbide a, as the substrate, were required for the formation of the precursor, and boiled proteins were enzymatically inactive. The maximum rate of conversion of pheophorbide a to the precursor occurred at pH 7.5. The K(m) for pheophorbide a was 12.5 μM at pH 7.0. Both pheophorbide b and bacteriopheophorbide a could serve as substrates, but protopheophorbide a could not. Formation of methanol was detected during the enzymatic reaction, an indication that the enzyme is an esterase. Among seven alcohol analogs tested, only methanol inhibited the enzymatic activity uncompetitively, with a K(i) of 71.6 mM. Mass-spectrometric (MS) analysis of the precursor yield a peak at m/z 579 that indicated the release of a methyl group from pheophorbide a. It appears therefore that the enzyme catalyzes the demethylation of the carbomethoxy group at C-132 of pheophorbide a by hydrolysis to yield methanol and the precursor, C-132-carboxyl-pyropheophorbide a, which is converted to pyropheophorbide a by spontaneous decarboxylation. We have tentatively designated the enzyme 'pheophorbidase. The presence of the enzyme was dependent on plant species and it was expressed constitutively.