L-Arginine kinase from tobacco hornworm, Manduca sexta (L.). Purification, properties, and interaction with L-canavanine.
Arginine kinase (adenosine 5'-triphosphate: L-arginine phosphotransferase, EC 2.7.3.3) was purified from the larvae of the tobacco hornworm, Manduca sexta (L). This enzyme catalyzes the production of L-phosphoarginine, which is the principal reserve of high energy phosphate compounds in insect muscle. The enzyme also phosphorylates L-canavanine, a guanidinooxy analogue of arginine which severely disrupts all developmental stages of this insect. Evaluations of certain kinetic and thermodynamic parameters of the reactions with arginine and canavanine suggest that reactions known to be much more sensitive to canavanine, such as protein synthesis or genome expression, rather than phosphagen formation and function account for the pronounced toxicity of canavanine in this insect. Sedimentation equilibrium and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate indicate that this insect enzyme has a molecular weight of about 40,000. This value is consistent with molecular weights of arginine kinases of non-insect arthropods. Its amino acid composition is also very similar to that of other arthropod arginine kinases. Km values for the enzyme are: L-arginine, 0.5 mM; Mg-ATP, 2.5 mM; L-canavanine, 22 mM; L-phosphoarginine, 0.7 mM; Mg-ADP, 0.45 mM; and L-phosphocanavanine, 27 mM. Turnover numbers (expressed as moles of product per min per mol of enzyme) are: L-arginine, 8,320; L-canavanine, 1,635; L-phosphoarginine, 25,875; and L-phosphocanavanine, 3,040. The apparent equilibrium constants at 37 degrees for phosphagen formation are 0.44 with arginine and 0.1 with canavanine. A procedure for L-phosphocanavanine synthesis is also presented.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002172:Canavanine / Q000378:metabolism*; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera / Q000201:enzymology*; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D002172:Canavanine; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases