Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate.
Maize malic enzyme was rapidly inactivated by micromolar concentrations of cupric nitrate in the presence of ascorbate at pH, 5.0. Ascorbate or Cu2+ alone had no effect on enzyme activity. The substrate L-malate or NADP individually provided almost total protection against Cu2+-ascorbate inactivation. The loss of enzyme activity was accompanied by cleavage of the enzyme. The cleaved peptides showed molecular mass of 55 kDa, 48 kDa, 38 kDa, and 14 kDa. Addition of EDTA, histidine and imidazole provided protection. The results of protection experiments with sodium azide, DABCO and catalase suggested that reactive oxygen species were generated resulting in loss of enzyme activity. This was further supported by experiments showing that the rate of enzyme inactivation was higher in D2O than in water. It is suggested that maize malic enzyme is modified by reactive oxygen species like singlet oxygen and H2O2 generated by Cu2+-ascorbate system and the modified amino acid residue(s) may be located at or near the substrate-binding site of the enzyme.
Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
S E
SE
Pinto
S R
SR
Rao
A S
AS
Bhagwat
D000596:Amino Acids / Q000737:chemistry; D001205:Ascorbic Acid / Q000737:chemistry*; D002374:Catalase / Q000737:chemistry; D003300:Copper / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D008293:Malates / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry; D017382:Reactive Oxygen Species; D003313:Zea mays / Q000378:metabolism*
D000596:Amino Acids; D007093:Imidazoles; D008293:Malates; D010879:Piperazines; D017382:Reactive Oxygen Species; D006639:Histidine; D009249:NADP; D003300:Copper; C029899:imidazole; C030298:malic acid; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D002374:Catalase; D017666:Deuterium Oxide; D001205:Ascorbic Acid; D010100:Oxygen; C007306:triethylenediamine