Purification and properties of a serine protease from Pseudomonas matophilia.
The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A see article; 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v)s20, w equal 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca2+, Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-L-tyrosine ethyl ester and alpha-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K1 was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0.
D000645:Ammonium Sulfate; D001152:Arsenicals / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors; D011549:Pseudomonas / Q000201:enzymology*; D012694:Serine; D012965:Sodium Chloride / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D001152:Arsenicals; D008670:Metals; D011480:Protease Inhibitors; D012965:Sodium Chloride; D012694:Serine; D004492:Edetic Acid; D010447:Peptide Hydrolases; D000645:Ammonium Sulfate; D002118:Calcium