Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase.
To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75 degrees C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.
Fernández-Murga
M Leonor
ML
Instituto de Biomedicina de Valencia (IBV-CSIC), C/ Jaime Roig 11, 46010-Valencia, Spain.
Gil-Ortiz
Fernando
F
Llácer
José L
JL
Rubio
Vicente
V
D001120:Arginine / Q000096:biosynthesis* / Q000494:pharmacology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000378:metabolism*
D003067:Coenzymes; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase