Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression

The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.     

Comparative fluorescence properties of lipoxygenases

1. Lipoxygenases purified from tomato, rat liver and soybean show a fluorescence band centered at 648 nm, which is likely to derive from Tyr and Trp. 2. The intensity of this fluorescence range from 0.7 to 1.0% of the intensity of their major intrinsic fluorescence band (lambda max = 343 nm) in all these lipoxygenases. 3. At inhibitory concentrations, ditizone partly quenches the fluorescence of the lipoxygenases above 600 nm. 4. Saturating concentrations of linoleic acid produce 79% quenching of the fluorescence at 648 nm of soybean lipoxygenase inactivated by treatment with 1 mM dithiothreitol. From these data we have obtained an apparent Kd for linoleic acid-lipoxygenase complex dissociation of 34 +/- 3 microM. 5. It is suggested that the fluorescence above 600 nm reveals the presence of aromatic amino acids located near or at the catalytic center.     

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant

The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.     

Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine alpha-amidating monooxygenase

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.     

Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

Substrate specificity of human and yeast aldehyde dehydrogenases

Human aldehyde dehydrogenase (ALDH) family may contribute to metabolism of hydrocarbons, biogenic amines, retinoids, steroids, and lipid peroxidation. We previously reported kinetic properties of human cytosolic ALDH1 and mitochondrial ALDH2 towards oxidation of the straight-chain and branched-chain aliphatic aldehydes with various chain lengths [S.J. Yin, M.F. Wang, C.L. Han, S.L. Wang, Substrate binding pocket structure of human aldehyde dehydrogenases: a substrate specificity approach, Adv. Exp. Med. Biol. 372 (1995) 9-16]. We present here substrate specificities for aromatic and heterocyclic aldehydes with purified human liver ALDH1 and ALDH2, and also with yeast mitochondrial ALDH2 for comparison. Kinetic assay for human ALDHs was performed in 50mM HEPES, pH 7.5 and 25 degrees C, containing 0.5mM NAD(+), 1.7% (v/v) acetonitrile (as a solvent carrier for aldehydes) and varied concentrations of substrate, and for yeast ALDH2 the assay was determined in the same reaction mixture except containing 3mM NAD(+) and addition of 200 mM KCl. With respect to phenylacetaldehyde, 2-phenylpropionaldehyde, benzaldehyde, p-nitrobenzaldehyde, cinnamaldehyde, 2-furaldehyde and indole-3-acetaldehyde, human liver ALDH1 exhibited K(M) ranging from 0.25 to 4.8 microM, V(max) of 0.34-2.4U/mg, and catalytic efficiency, V(max)/K(M), 0.070-3.9U/(mg microM); human ALDH2 exhibited K(M) ranging from less than 0.15-0.74 microM, V(max) of 0.039-0.51 U/mg, and V(max)/K(M), 0.15-1.0U/(mg microM). Human ALDH1 and ALDH2 exhibited substate inhibition constants (K(i)) for phenylacetaldehyde, 95 and 430 microM, respectively. Yeast ALDH2 exhibited K(M) for straight-chain aliphatic aldehydes (C1-C10), 2.3-210 microM, and substrate inhibition constants (C2-C10), 79-2900 microM, with a trend of being smaller K(M) and K(i) for longer chain lengths; and K(M) for cinnamaldehyde, benzaldehyde, and 2-furaldehyde, 5.0, 79, and 1000 microM, respectively. Therefore human ALDH1/ALDH2 and yeast ALDH2 can contribute to detoxification or metabolism of various exogenous/endogenous aliphatic and aromatic aldehydes. The systematic changes in kinetic parameters for oxidation of structurally related aldehydes may reflect subtle functional topographic distinctions of substrate pocket for human and yeast ALDHs.     

Induced fit and kinetic mechanism of adenylation catalyzed by Escherichia coli threonyl-tRNA synthetase

Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.     

Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.     

Expression and characterization of frog peptidylglycine alpha-hydroxylating monooxygenase

We report here a recombinant Chinese hamster ovary cell system,which is able to stably express frog peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3), the first enzyme responsible for the formation of peptide C-terminal amide. This system excreted PHM mostly into the medium and almost no PHM activity was detected in the cell lysate. Three differentiation inducers were examined to determine whether or not they would enhance the PHM expression. Addition of 4mM sodium butyrate into the medium increased the expression of PHM activity about 4-fold at 48 h after addition. Increases of about 2-fold were observed in the cases of sodium propionate or N,N(')-hexamethylene-bis-acetamide. Through a three-step purification procedure, we obtained 5mg purified PHM, which showed a single band at 40 kDa on SDS-PAGE, from 2-L of conventional monolayer culture medium. The reactions with three synthetic substrates, D-Tyr-Val-Gly, N-trinitrophenyl-D-Tyr-Val-Gly (TNPYVG), and hippuric acid (HA), were characterized. Of these, TNPYVG was the most active substrate. The pH optima for TNPYVG and HA were pH 5-6, while that for D-Tyr-Val-Gly was pH 7.5. There is a possibility that the substrate N-terminal structure may affect the interaction between the substrate and the enzyme catalytic site.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-beta-D-cellobioside

A simple and reliable continuous assay procedure for measurement of cellulase activity from several species using the new substrate resorufin-beta-D-cellobioside (Res-CB) has been developed. The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, which allows continuous measurement of fluorescence turnover at or near physiological pH values. The assay performed using purified cellulase from the microscopic fungus Trichoderma reesei has been shown to give the kinetic parameters K(m) of 112 microM and V(max) of 0.000673 micromol/mL/min. Methods for performing the assay using cellulases isolated from both live Arabidopsis thaliana plant and Aspergillus niger fungal species are presented.     

Stopped-flow fluorescence studies of inhibitor binding to tyrosinase from Streptomyces antibioticus

Tyrosinase (Ty) is a type 3 copper protein involved in the rate-limiting step of melanin synthesis. It is shown that the endogenous Trp fluorescence of tyrosinase from Streptomyces antibioticus is remarkably sensitive to the redox state. The fluorescence emission intensity of the [(Cu(I) Cu(I)] reduced species is more than twice that of the oxygen-bound [Cu(II)-O(2)(2-)-Cu(II)] form. The emission intensity of the oxidized [Cu(II)-OH(-)-Cu(II)] protein (Ty(met)) appears to be dependent on an acid-base equilibrium with a pK(a) value of 4.5 +/- 0.1. The binding of fluoride was studied under pseudo first-order conditions using stopped-flow fluorescence spectroscopy. The kinetic parameters k(on), K(d), and the fraction of fluorescence emission quenched upon fluoride binding show a similar pH dependence as above with an average pK(a) value of 4.62 +/- 0.05. Both observations are related to the dissociation of Cu(2)-bridging hydroxide at low pH. It is further shown that Ty is rapidly inactivated at low pH and that halide protects the enzyme from this inactivation. All results support the hypothesis that halide displaces hydroxide as the Cu(2)-bridging ligand in Ty(met). The relevance of the experimental findings for the catalytic cycle is discussed. The data are consistent with the data obtained from other techniques, validating the use of fluorescence quenching as a sensitive and effective tool in studying ligand binding and substrate conversion.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.     

Purification and characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.     

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.     

Fluorometric study of the acid-induced denaturation of Staphylococcal nuclease and its mutant forms

The acid-induced denaturation of wild-type Staphylococcal nuclease (WT) and its eight mutant forms, L25A, V66L, G79S, A90S, G88V, H124L, V66L/G88V, and V66L/G79S/G88V, was investigated using Trp140 fluorescence as a probe at 30 degrees C. The values of pH(1/2), at which the denaturation is half completed, and n, the apparent number of protons which trigger the denaturation and are taken up by the proteins upon denaturation at pH(1/2), were evaluated from the pH dependence of the fluorescence intensity. The values of pH(1/2) and n for WT were 3.8 and 1.8 respectively. The amino acid replacements changed the pH(1/2) values to a range between 3.0 (H124L) and 4.4 (G79S) and also changed the n values to a range between 1.0 (A90S) and 3.0 (G88V). There was a negative correlation between the values of pH(1/2) and n. It was suggested that the amino acid replacements may change the energy levels of the native state and/or the denatured state mainly in the neutral (stable) pH region, not in the acidic (unstable) region, resulting in the correlative changes in pH(1/2) and n.     

Fluorescence-based continuous assay for the aspartyl protease of human immunodeficiency virus-1

5-Dimethylaminoaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-T rp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (lambda ex 290 nm, lambda em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations less than 60 microM, initial reaction velocity increased as a linear function of substrate concentration, with kcat/KM = 9700 M-1 s-1. Limited solubility and internal fluorescence quenching precluded a determination of KM for Dns-SQNYPIVW, but this was clearly greater than 100 microM.     

Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression

Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V(max) and K(m) values of 0.0551 microM/min and 234 microM, respectively, and the substrate DL-Val-Leu-Arg-pNA with V(max) and K(m) values of 0.3830 microM/min and 429 microM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23C. Proteolytic activity was reduced by 90% at 60 degrees C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.     

Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.     

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.