Human protoporphyrinogen oxidase: relation between the herbicide binding site and the flavin cofactor.
Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants. Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships. A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-[3H]tetrahydrophthalimide ([3H]AzTHP), resulting in high specific activity (2.6 TBq/mmol). Human protox expressed in Escherichia coli and purified by affinity chromatography is used with [3H]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [3H]AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The Bmax establishes one [3H]AzTHP binding site per FAD. Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting [3H]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD. The first step has been taken in photoaffinity labeling the herbicide/substrate site with [3H]AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.
Birchfield
N B
NB
Department of Environmental Science, University of California, Berkeley 94720-3112, USA.
Latli
B
B
Casida
J E
JE
D001665:Binding Sites; D018548:Cotyledon / Q000187:drug effects / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D005420:Flavoproteins; D006540:Herbicides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D024101:Mitochondrial Proteins; D009861:Onium Compounds / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D019920:Photoaffinity Labels / Q000378:metabolism*; D010797:Phthalimides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D050768:Protoporphyrinogen Oxidase; D013329:Structure-Activity Relationship; D014316:Tritium
D005415:Flavins; D005420:Flavoproteins; D006540:Herbicides; D024101:Mitochondrial Proteins; C112222:N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5,6-tetrahydrophthalimide; D009861:Onium Compounds; D019920:Photoaffinity Labels; D010797:Phthalimides; D014316:Tritium; D005182:Flavin-Adenine Dinucleotide; C007517:diphenyleneiodonium; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase