Crystallization and properties of L-arginine deiminase of Pseudomonas putida.
Crystalline L-arginine deiminase of Pseudomonas putida was prepared by the following steps: sonic disruption, ammonium sulfate fractionation, protamine sulfate treatment, DEAE-cellulose column chromatography, and L-arginine-Sepharose 6B chromatography followed by crystallization. This procedure yields a crystalline pure enzyme with a 45% recovery of the activity in crude cell-free extracts. The yield is significantly higher than that reported for this enzyme. The purified enzyme appears to be homogeneous in ultracentrifugation (s-o20, w equals 10.2 S) and isoelectric focusing (pI equals 6.13). The purified enzyme showed two bands on disc gel electrophoresis, both carrying out the deimination of L-arginine. Electrophoresis in the presence of beta-mercaptoethanol plus Na dodecyl-SO4 gave a single band (Mr, 54,000). Specific activity of this enzyme was 58.8 mumol of L-citrulline formed per min per mg of protein at 37 degrees. The optimum pH of the purified enzyme was 6.0 and maximal activity was obtained at 50 degrees. The molecular weight of the native protein was 130,000 by gel filtration and 120,000 by sedimentation-equilibrium measurements. The spectrum of the pure enzyme showed absorption maximum at 280 nm and the value of E-1%-1 CM AT 280 NM WAS 10.48 IN 0.05 M potassium phosphate buffer (pH 7.0). The crystalline enzyme hydrolyzed several L-arginine analogues. L-Homoarginine, L-alpha-amino-gamma-guanidinobutyric acid, and L-alpha-amino-beta-guanidinopropionic acid competitively inhibited the hydrolysis of L-arginine with Ki values of 25.7, 7.5, and 4.0 times 10- minus 3 M, respectively. p-Chloromercuribenzoate, Ag-+, and Hg-2+, and several metal ions inhibited the enzyme.
D001120:Arginine; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011479:Protamines; D011549:Pseudomonas / Q000201:enzymology*; D013045:Species Specificity; D013696:Temperature
D002413:Cations, Divalent; D002414:Cations, Monovalent; D011479:Protamines; D001120:Arginine; D006867:Hydrolases