ENZYMATIC CONVERSION OF PHEOPHORBIDE A TO THE PRECURSOR OF PYROPHEOPHORBIDE A IN LEAVES OF CHENOPODIUM-ALBUM

Soluble proteins extracted from leaves of Chenopodium album catalyzed the conversion of pheophorbide a to a precursor of pyropheophorbide a, putatively identified as C-13(2)-carboxyl-pyropheophorbide a. The pre cursor was then decarboxylated non-enzymatically to yield pyropheophor bide a, Soluble proteins and pheophorbide a, as the substrate, were re quired for the formation of the precusor, and boiled proteins were enz ymatically 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 mu M at pH 7.0. Both pheophorbide b and bacteriopheophorbide a coul d serve as substrates, but protopheophorbide a could not. Formation of methanol was detected during the enzymatic reaction, an indication th at 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 phe ophorbide a. It appears therefore that the enzyme catalyzes the demeth ylation of the carbomethoxy group at C-132 of pheophorbide a by hydrol ysis to yield methanol and the precursor, C-13(2)-carboxyl-pyropheopho rbide a, which is converted to pyropheophorbide a by spontaneous decar boxylation. We have tentatively designated the enzyme ''pheophorbidase ''. The presence of the enzyme was dependent on plant species and it w as expressed constitutively.