Studies on N-acetylneuraminic acid aldolase

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.     

The reaction of N-acetylneuraminate lyase with chloropyruvate (Short Communication)

Chloropyruvate, like bromopyruvate, rapidly inactivates N-acetylneuraminate lyase at pH7.2. At 5 degrees C, 0.5mm-chloropyruvate reacted with the enzyme about ten times as fast as bromopyruvate. In contrast, at pH6.0 and 9 degrees C, chloropyruvate reacted with N-acetylcysteine seven times more slowly than bromopyruvate. A brief (2min) incubation of the enzyme with 1.0mm-chloro[(14)C]pyruvate gave an inactive enzyme in which 4.5 cysteine residues were alkylated per molecule of enzyme. This corresponded to the number of [(14)C]pyruvate residues (3.7) bound to the enzyme by borohydride reduction of the [(14)C]-pyruvate complex, and confirmed the previous suggestion that there is one ;essential' cysteine residue per active site. It is suggested that, for this enzyme, chloropyruvate can be selectively used to alkylate the active-site residues, whereas bromopyruvate cannot. The apparent molecular weight of the enzyme prepared by two slightly different methods was approx. 100000 or 250000. The latter value has been used to calculate the number of pyruvate residues bound to the enzyme.     

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.     

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.     

Phosphoenolpyruvate carboxylase of Escherichia coli. Affinity labeling with bromopyruvate.

Phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromopyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkylation. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyruvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5,5'-dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. The results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent.     

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.     

Bromopyruvate inactivation of 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila. Kinetics and stereochemistry.

The enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila is inactivated by the substrate analog beta-bromopyruvate, which satisfies several criteria of being an active site directed reagent. The inactivation exhibits saturation kinetics, and both bromopyruvate and pyruvate (substrate) compete for free enzyme. Upon prolonged incubation, inactivation is virtually complete. The Kinact for bromopyruvate is 12 mM and the minimum inactivation half-time is 16 min with a k of 0.0433 min minus 1. Bromopyruvate is also a substrate for the enzyme in that 3(R,S)-[3-3H2]bromopyruvate is asymmetrically detritiated by the enzyme yielding 3(S)-[3-3H,H]bromopyruvate concomitant with inactivation. At various concentrations of bromopyruvate which affect the inactivation rate, the ratio of nanomoles of bromopyruvate turned over/unit of enzyme inactivated remains constant averaging 12:1, consistent with both inactivation and catalysis occurring at a single protein site, the catalytic site. The above value does not take into account a possible hydrogen isotope effect and is not thus an absolute value. The stereochemistry of bromopyruvate turnover catalyzed by this enzyme is the same as that for 2-keto-3-deoxy-6-phosphogluconate aldolase of P. putida. This fact provides the first evidence that the pyruvate-specific portions of the two active sites may have evolved from a common precursor.     

Bromopyruvate inactivation of glutamate apodecarboxylase. Kinetics and specificity.

A number of halo carboxylic and dicarboxylic acids were substrate-competitive inhibitors of glutamate decarboxylase, with bromosuccinate, 3-bromopropionate, and iodoacetate having the highest affinity for the enzyme. Some of the halo acids also inactivated the apoenzyme. Bromopyruvate at relatively low concentrations inactivated the apoenzyme irreversibly. The rate of the inactivation of the apodecarboxylase was proportional to bromopyruvate at low concentration and approached a constant rate of inactivation at high bromopyruvate concentration. These data are consistent with a two-step inactivation process in which an enzyme-bromopyruvate complex is formed followed by inactivation. The concentration of bromopyruvate giving the half-maximum rate of inactivation was 6.9 mM, and the maximum rate of inactivation was 1.75 min-1 at pH 4.6 and 23 degrees. Much faster rates of inactivation were obtained at pH 5.96 and 6.44. Phosphate, an inhibitor of pyrisoxal-P binding to the apoenzyme, competitively inhibited the inactivation of the apoenzyme by bromopyruvate. In addition, bromopyruvate inhibited the rate of pyridoxal-P binding to the apoenzyme. Kinetics of the incorporation of bromo[2-14C]pyruvate indicated that complete inactivation was obtained when 1.2 mol of radioactive residue were covalently bound per subunit of apoenzyme. Amino acid analyses demonstrated that a cysteinyl residue was alkylated by the bromopyruvate. The bromopyruvate was evidently interacting nincovalently with a cationic group at or near the pyridoxal-P-binding site, and then was alkylating a nearby cysteinyl residue.     

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.     

N-linked glycosylation of a beetle (Apriona germari) cellulase Ag-EGase II is necessary for enzymatic activity

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family (GHF) 45 of the mulberry longicorn beetle, Apriona germari (Ag-EGase II), has three potential N-linked glycosylation sites; these sites are located at amino acid residues 56-59 (NKSG), 99-102 (NSTF), and 237-239 (NYSstop). In the present study, we analyze the functional role of these potential N-linked glycosylation sites. Tunicamycin treatment completely abolished the enzymatic activity of Ag-EGase II. To further elucidate the functional role of the N-linked glycosylation sites in Ag-EGase II, we have assayed the cellulase enzyme activity in Ser58Gln, Thr101Gln, or Ser239Gln mutants. Lack of N-linked glycosylation site at residues 99-102 (NSTF), the site of which is conserved in known beetle GHF 45 cellulases, showed loss of enzyme activity and reduced the molecular mass of the enzyme. In contrast, mutations in Ser58Gln or Ser239Gln affected neither the activity nor the apparent molecular mass of the enzyme, indicating that these sites did not lead to N-linked glycosylation. The present study demonstrates that N-linked glycosylation at residues 99-102 (NSTF), while not essential for secretion, is required for Ag-EGase II enzyme activity.     

5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction

Bone sialoprotein (BSP) is an anionic phosphorylated glycoprotein that is expressed almost exclusively in mineralized tissues and has been shown to be a potent nucleator of hydroxyapatite formation. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and in the adhesion of bone cells to the mineralized matrix. Using a solid phase assay, we have investigated the interaction between BSP and collagen. Initial studies showed that raising the ionic strength, decreasing the pH below 7, or introducing divalent cations diminishes but does not abolish the binding of BSP to collagen, indicating that the interaction is only partly electrostatic in nature. Both bone-extracted and recombinant (r)BSP exhibited similar binding affinities, indicating that post-translational modifications are not critical for binding. To identify the collagen-binding domain, recombinant peptides of BSP were studied. Peptide rBSP-(1-100) binds to type I collagen with an affinity similar to that of full-length rBSP, whereas peptides containing the sequences 99-201 or 200-301 do not bind. Further studies showed that rBSP-(1-75) competitively inhibits the binding of rBSP-(1-100), whereas rBSP-(21-100) inhibits binding to a lesser extent, and rBSP-(43-100) does not inhibit binding. These results suggest that the collagen-binding site of rat BSP is within the sequence 21-42, with residues N-terminal of this region likely also involved. This site was confirmed by the demonstration of collagen-binding activity of a synthetic peptide corresponding to residues 19-46. The collagen-binding domain, which is highly conserved among species, is enriched in hydrophobic residues and lacks acidic residues. We conclude that residues 19-46 of BSP represent a novel collagen-binding site.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.     

Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis

The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.     

Modification of the cysteine residues of cytochrome P-450cam with 2-bromoacetamido-4-nitrophenol

The Pseudomonas putida cytochrome P-450 was alkylated with the SH-reagent, 2-bromoacetamido-4-nitrophenol. One out of eight cysteine residues present in the enzyme reacted rapidly while another 3 ～ 4 cysteine residues were gradually alkylated at longer reaction times. The derivative in which the most reactive cysteine residue was labeled with this reagent was hydrolyzed with trypsin and a tryptic peptide isolated. From the amino acid composition and end group analysis of the peptide, the rapidly reacting cysteine residue was shown to be Cys 355. This cysteine residue is probably exposed on the surface and is involved in the dimerization of the enzyme. The amino acid sequence about cysteine 355 shows sequence homology with residues 429–445 of the rat liver cytochrome P-450-LM-2.     

Differential modification of specificity in carbonic anhydrase catalysis

Incubation of carbonic anhydrase II with acrolein results in a rapid, time-dependent loss of all but approximately 3-6% of the original catalytic activity toward CO2 hydration and HCO3- dehydration, with the inactivation rate being first-order in both acrolein and the enzyme. The pH dependence of the inactivation rate constant can be adequately described with a function incorporating a pK alpha of 7.15 and a maximal value for kinact [corrected] of 26.2 M-1 min-1, indicating that at least one of the catalytically essential residues that ionizes at this pH is involved in the modification scheme. The amount of residual CO2 hydratase activity is proportional to the molar excess of acrolein over carbonic anhydrase II with 5 histidyl and 3 lysyl residues being subject to alkylation under conditions where [acrolein] to [carbonic anhydrase II] ratio is greater than 100. Because all lysyl residues were shown previously to be amidinated without detectable loss of activity, it was assumed that the modification of one (or more) of the histidines was primarily responsible for the observed inactivation. The number of modified histidyl residues could be related to residual activity by using the statistical analysis of Tsou (Tsou, C.-L. (1962) Sci. Sin. (Engl. Ed.) 11, 1535-1558) which indicates that one essential histidine reacts approximately four times faster than the other (histidyl) residues. In sharp contrast with the phenomenon observed in connection with CO2 hydration and HCO3- dehydration, acrolein improves the catalytic efficiency of the enzyme toward p-nitrophenyl acetate hydrolysis and acetaldehyde hydration, with the relative activity increasing by approximately 12 and 34%, respectively. The widely differing effects imparted by the same reagent represent the first step toward differential control of the specificity of carbonic anhydrase II.     

cDNA cloning,high-level expression,purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck

As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing. By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver. Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature. Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS. After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained. Duck SOD is a homodimer with 153 residues for each subunit. The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart. Five charge isomers were observed on isoelectricfocusing (IEF). The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C. These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.     

Identification of the G protein-activating domain of the natriuretic peptide clearance receptor (NPR-C).

We have shown recently that the 37-amino acid intracellular domain of the single-transmembrane, natriuretic peptide clearance receptor, NPR-C, which is devoid of kinase and guanylyl cyclase activities, activates selectively Gi1 and Gi2 in gastric and tenia coli smooth muscle. In this study, we have used synthetic peptide fragments of the N-terminal, C-terminal, and middle regions of the cytoplasmic domain of NPR-C to identify the G protein-activating sequence. A 17-amino acid peptide of the middle region (Arg469-Arg485), denoted Peptide 4, which possesses two N-terminal arginine residues and a C-terminal B-B-X-X-B motif (where B and X are basic and non-basic residues, respectively) bound selectively to Gi1 and Gi2, activated phospholipase C-beta3 via the betagamma subunits, inhibited adenylyl cyclase, and induced smooth muscle contraction, in similar fashion to the selective NPR-C ligand, cANP4-23. A similar sequence (Peptide 3), but with a partial C-terminal motif, had minimal activity. Sequences which possessed either the N-terminal basic residues (Peptide 1) or the C-terminal B-B-X-X-B motif (Peptide 2) were inactive. Peptide 2, however, inhibited G protein activation and cellular responses mediated by the stimulatory Peptide 4 and by cANP4-23, suggesting that the B-B-X-X-B motif mediated binding but not activation of G protein, thus causing Peptide 2 to act as a competitive inhibitor of G protein activation.     

Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. Bromopyruvate inactivation and labeling of glutamate 45

Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity. Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate. Inactivation studies with [14C] bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45. Incubation of the enzyme with excess [14C]bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well. Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues. The results indicate that Glu-45 of E. coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed.