Inhibition kinetics of cabbage butterfly (Pieris rapae L.) larvae phenoloxidase activity by 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone.
Phenoloxidase (PO) is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from Pieris rapae (Lepidoptera) larvae in the oxidation of L-tyrosine (a monophenol) and L-DOPA (l-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) on the monophenolase and diphenolase activities of PO were also studied. The results show that 3-H-4-MBT can inhibit both the monophenolase and diphenolase activities of PO. The lag period of L-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased. The inhibitor was found to be noncompetitively reversible with a K I (K I = K IS) of 0.30 micromol/L and an estimated IC50 of 0.14 +/- 0.02 micromol/L for monophenolase and 0.26 +/- 0.04 micromol/L for diphenolase. In the time course of the oxidation of L-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants for the reaction of 3-H-4-MBT with the enzyme were determined.
Xue
Chao-Bin
CB
College of Plant Protection, Key Laboratory of Pesticide Toxicology and Application Technique, Shandong Agricultural University, Shandong Tai'an, People's Republic of China.
Luo
Wan-Chun
WC
Jiang
Lin
L
Xie
Xian-Ye
XY
Xiao
Ting
T
Yan
Lei
L
D000818:Animals; D002080:Butterflies / Q000201:enzymology*; D006139:Guaiacol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D007980:Levodopa / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013882:Thiosemicarbazones / Q000737:chemistry / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
C525512:3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone; D013882:Thiosemicarbazones; D014443:Tyrosine; D007980:Levodopa; D006139:Guaiacol; D010088:Oxidoreductases; C412191:monophenolase; D014442:Monophenol Monooxygenase