Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a K_m for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.     

High resolution crystal structure of a Mg2+-dependent porphobilinogen synthase.

Common to the biosynthesis of all known tetrapyrroles is the condensation of two molecules of 5-aminolevulinic acid to the pyrrole porphobilinogen catalyzed by the enzyme porphobilinogen synthase (PBGS). Two major classes of PBGS are known. Zn2+-dependent PBGSs are found in mammals, yeast and some bacteria including Escherichia coli, while Mg2+-dependent PBGSs are present mainly in plants and other bacteria. The crystal structure of the Mg2+-dependent PBGS from the human pathogen Pseudomonas aeruginosa in complex with the competitive inhibitor levulinic acid (LA) solved at 1.67 A resolution shows a homooctameric enzyme that consists of four asymmetric dimers. The monomers in each dimer differ from each other by having a "closed" and an "open" active site pocket. In the closed subunit, the active site is completely shielded from solvent by a well-defined lid that is partially disordered in the open subunit. A single molecule of LA binds to a mainly hydrophobic pocket in each monomer where it is covalently attached via a Schiff base to an active site lysine residue. Whereas no metal ions are found in the active site of both monomers, a single well-defined and highly hydrated Mg2+is present only in the closed form about 14 A away from the Schiff base forming nitrogen atom of the active site lysine. We conclude that the observed differences in the active sites of both monomers might be induced by Mg2+-binding to this remote site and propose a structure-based mechanism for this allosteric Mg2+in rate enhancement.     

Phospholipase C from Clostridium novyi type A. II. Factors influencing the enzyme activity

The apparent activity of phospholipase C[EC 3.1.4.3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates. When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5 mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5 mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation. The isoelectric point of the enzyme was 7.1 +/- 0.1.     

Substrate-induced interconversion of protein quaternary structure isoforms

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

金属离子对蜡蚧菌几丁质酶活力的影响

以蜡蚧菌(Ll)发酵液为材料,经分离纯化获得Ll几丁质酶(EC3.2.1.14)制剂.研究了金属离子对Ll几丁质酶活力的影响.结果表明,K+、Mg2+、Zn2+、Ca2+和Fe3+对几丁质酶活性有明显的促进作用,而Na+和Cu2+完全抑制几丁质酶的活性;Mn2+在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;Fe2+和Ba2+的浓度低于0.5 mmol/L时对酶起抑制作用,而高于该浓度时则对酶有激活作用.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.     

Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.     

Tracking the evolution of porphobilinogen synthase metal dependence in vitro

Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.     

Purification and some properties of ATP-sulfurylase from developing sea urchin embryos

ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4), purified about 200-fold from sea urchin embryos, was free of ATPase and inorganic pyrophosphatase. The molecular weight of the enzyme was approx. 280 000 measured by gel filtration. The enzyme was activated by Mg2+, Ca2+ or Zn2+; EDTA and p-chloromercuriphenylsulfonate inhibited the enzyme activity. The inhibition was reversed by addition of Mg2+ and dithiothreitol, respectively. The enzyme activity increased continuously as the pH was raised from 5.6 to 10.6. The Km values for the enzyme were calculated to be 13 microM for adenosine 5'-phosphosulfate and 23 microM for pyrophosphate.     

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.