Transpeptidation reactions of a specific substrate catalyzed by the Streptomyces R61 DD-peptidase: the structural basis of acyl acceptor specificity

Bacterial dd-peptidases, the targets of beta-lactam antibiotics, are believed to catalyze d-alanyl-d-alanine carboxypeptidase and transpeptidase reactions in vivo. To date, however, there have been few concerted attempts to explore the kinetic and thermodynamic specificities of the active sites of these enzymes. We have shown that the peptidoglycan-mimetic peptide, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, 1, is a very specific and reactive carboxypeptidase substrate of the Streptomyces R61 dd-peptidase [Anderson, J. W., and Pratt, R. F. (2000) Biochemistry 39, 12200-12209]. In the present paper, we explore the transpeptidation reactions of this substrate, where the enzyme catalyzes transfer of the glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl moiety to amines. These reactions are believed to occur through capture of an acyl-enzyme intermediate by amines rather than water. Experiments show that effective acyl acceptors require a carboxylate group and thus are amino acids and peptides. d(but not l)-amino acids, analogues of the leaving group of 1, are good acceptors. The effectiveness of d-alanine as an acceptor increases with pH, suggesting that the bound and reactive form of an amino acid acceptor is the free amine. Certain glycyl-l(but not d)-amino acids, such as glycyl-l-alanine and glycyl-l-phenylalanine, are also good acceptors. These molecules may resemble the N-terminus of the Streptomyces stem peptides that, presumably, are the acceptors in vivo. The acyl acceptor binding site therefore demonstrates a dual specificity. That d-alanyl-l-alanine shows little activity as an acceptor suggested that, on binding of acceptors to the enzyme, the carboxylate of d-amino acids does not overlap with the peptide carbonyl group of glycyl-l-amino acids. Molecular modeling of transpeptidation tetrahedral intermediates and products demonstrated the likely structural bases for the stereospecificity of the acceptors and the nature of the dual function acceptor binding site. For both groups of acceptors, the terminal carboxylate appeared to be anchored at the active site by interaction with Arg 285 and Thr 299.     

PARTIAL PURIFICATION AND KINETICS OF γ-GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS

Abstract— γ-Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane-bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L-Methionine was the best amino acid substrate and its corresponding peptides L-methionylmethionine and L-methionyl-L-serine were also good γ-glutamyl acceptors. L-Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ-glutamyl acceptor than glycine, triglycine, or tetraglycine. L-Alanylglycine was a superior acceptor to glycine, L-alanine, or L-alanylglycylglycine, while the D-isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ-glutamyl transpeptidase could catalyze the transfer of γ-glutamyl groups to glycylglycyl-L-alanine and L-alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ-glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.     

The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney gamma-glutamyltransferase

The effect of pH upon the transpeptidation and hydrolytic reactions of gamma-glutamyltransferase [5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2) have been investigated. It was found that the enzyme was irreversibly inactivated below pH 7.5 or above pH 9.4. Transpeptidation was markedly pH-dependent, while hydrolysis was pH-independent. The pH optimum for transpeptidation was found to vary for different acceptors. The ascending limb of the pH-optimum curve is attributed to the pK of the alpha-amino group of the acceptor, while the descending limb of the pH-optimum curve is attributed to an ionisable group in the active site of the enzyme. These observations provide much information about the interaction of the enzyme with the acceptor: (1) the true acceptor for gamma-glutamyltransferase is the deprotonated form of the amino acid; (2) glycylglycine has a similar acceptor activity to methionine, its apparent higher activity being due to the low pK of the alpha-amino group; (3) the enzyme is reversibly inactivated at higher pH by the deprotonation of a group in the active site which is involved in both binding of acceptor and catalysis of transpeptidation (this group is not involved in the hydrolysis reaction); (4) at pH 8.5, the normal pH for assay, only 47% of the enzyme is active, while at pH 7.4 gamma-glutamyltransferase is 93% in the active form.     

Hydrogen bonding of iron-coordinated histidine in heme proteins.

The hydrogen-bonding motifs of the proton on the N delta atom of iron-coordinated histidine residues in heme proteins have been classified into three categories: (1) Those in which the hydrogen-bond acceptor is either an amino acid residue (serine) directly adjacent to the histidine or a carbonyl group of the polypeptide chain less than five residues away from the histidine; (2) those in which the hydrogen-bonding acceptor is a carbonyl group of the polypeptide backbone associated with an amino acid residue 8 to 17 residues away from the histidine; and (3) those in which the hydrogen-bonding acceptor is an exogenous water molecule or an amino acid residue located far from the histidine in the amino acid sequence. Some biological functions are defined by this classification, whereas others span all classes.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Synthesis of peptides containing oxo amino acids and their crystallographic analysis

An isolated uncharged hydrogen bond acceptor such as the carbonyl functionality of an aldehyde or a keto group is absent in natural amino acids. Although glutamine and asparagine are known to hydrogen bond through the amide carbonyl group in their side chains, they also possess the amide ? NH₂ group, which can act as a hydrogen bond donor. This makes the structural study of peptides containing an oxo residue, with an isolated carbonyl group in the side chain, interesting. Here, we report the synthesis of δ‐ and ε‐oxo amino acids and their incorporation into oligopeptides as the N‐terminal residue. The resultant oxo peptides were extensively studied using X‐ray crystallography to understand the interactions offered by the oxo group in peptide crystals. We find that the oxo groups are capable of providing additional hydrogen bonding opportunities to the peptides, resulting in increased intermolecular interactions in crystals. The study thus offers avenues for the utilization of oxo residues to introduce intermolecular interactions in synthetic peptides.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Electronic properties of the amino acid side chains contribute to the structural preferences in protein folding.

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: alpha-helix, beta-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (esigma), and a composite of these two scales (epsilon). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for alpha-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

The structure of cat muscle pyruvate kinase.

The complete amino acid sequence of cat muscle pyruvate kinase has been determined and fitted to the 2.6 A resolution electron density map. Residues in the active site region are highly conserved in the cat muscle, chicken muscle, rat liver and yeast enzymes. The enzyme-bound magnesium, which is essential for activity, interacts with the side chain of glutamate-271 and with two main carbonyl groups. Lysine-269 is the probable acid/base catalyst responsible for the interconversion of pyruvate and enolpyruvate. A possible binding site for the essential monovalent cation is proposed.     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

The mechanism of action of dipeptidyl aminopeptidase. Inhibition by amino acid derivatives and amines; activation by aromatic compounds.

A variety of amino acid and peptide amides have been shown to be inhibitors of dipeptidyl aminopeptidase. Among these compounds derivatives of strongly hydrophobic amino acids are the strongest inhibitors (Phe-NH2, Ki = 1.0 +/- 0.2 mM), while amides of basic amino acids were somewhat less effective (Lys-NH2, Ki = 36 +/- 3 mM). Short chain amino acid amides are notably weaker inhibitors (Gly-NH2, Ki = 293 +/- 50 mM). The interaction of the side chains of compounds with the enzyme appears to be at a site other than that at which the side chain of the amino-penultimate residue of the substrate interacts since the specificity of binding is different. Primary amines have been shown to inhibit, e.g., butylamine, Ki = 340 +/- 40 mM, and aromatic compounds have been shown to stimulate activity toward Gly-Gly-NH2 and Gly-Gly-OEt (phenol, 35% stimulation of activity at a 1:1 molar ratio with the substrate). The data suggest that inhibition involves binding at the site occupied by the free alpha-amino group and the N-terminal amino acid.     

Electronic Properties of the Amino Acid Side Chains Contribute to the Structural Preferences in Protein Folding

Abstract

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: α-helix, β-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (eσ), and a composite of these two scales (ε). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for α-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.     

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.     

Purification and some molecular properties of protein methylase II from equine erythrocytes

Protein methylase II (S-adenosyl-L-methionine: protein carboxyl-O-methyltransferase; E.C. 2.1.1.24) has been purified 28 000 fold from equine erythrocytes. The purified enzyme is homogeneous on polyacrylamide gel electrophoresis performed either in presence or in absence of SDS, and on analytical ultracentrifugation. It appears constituted of a single polypeptidic chain of a molecular weight very close to 25 000 Daltons. Other enzymatic properties of the protein are quite similar to those previously reported for similar enzymes. The amino acid analysis of the enzyme is presented. The single cysteine residue, the enzyme contains, is essential for the enzymatic activity. Other amino acids apparentely involved in catalysis are tentatively identified.     

Purification and characterization of a carbohydrate: acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently

A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized beta-(1-->4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the beta-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.     

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1

Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.     

Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts.

The catalytical role of the hydroxy amino acid in the "marker sequence" Asn-Xaa-Thr(Ser) for the N-glycosylation step of glycoprotein formation was investigated by using a series of hexapeptides derived from Tyr-Asn-Gly-Xaa-Ser-Val by substituting threonine, serine, cysteine, valine and O-methylthreonine respectively for Xaa. The results, which were obtained with calf liver microsomal fractions as enzyme source and dolichyl diphosphate di-N-acetyl [14C] chitobiose as glycosyl donor showed that the threonine-, serine- and cysteine-containing derivatives could be glycosylated, although at very different rates, whereas the valine and O-methylthreonine analogues did not work as glycosyl acceptors. Replacement of threonine by serine resulted in a 4-fold decrease in Vmax, and about a 10-fold increase in Km for glycosyl transfer. Replacement of serine by cysteine again decreased acceptor activity 2-3-fold. The various results, taken together, indicate an absolute requirement for a hydrogen-bond-donor function in the side chain of the hydroxy amino acid of the "marker sequence" and furthermore, point to a considerable influence of the structure of this amino acid on binding as well as on the glycosyl transfer itself. In order to explain the observed differences in the glycosyl-transfer rates, a model is proposed with a hydrogen-bond interaction between the amide of asparagine as the hydrogen-bond donor and the oxygen of the hydroxy group of the hydroxy amino acid as the hydrogen-bond acceptor. The participation of the hydroxy group in the catalytic mechanism of glycosyl transfer in the kind of proton-relay system is discussed.     

Purification and characterization of gamma-glutamyltransferase from rat pancreas

gamma-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-gamma-glutamyl-p-nitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylglycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney gamma-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.     

酸水解蚕蛹制备复合氨基酸的研究

采用硫酸水解法,以蚕蛹制取复合氨基酸产品,得到氨基酸态氮分别为９．０５％和１３．４５％的食用复合氨基粉和精制复合氨基酸粉。食用复合氨基酸粉含有１８种氨基酸,其中必需氨基酸含量为３９．２％。食用复合氨基酸粉的制备方法经工厂小批量生产证实,其工艺简单易行,适合于中小企业采用,该产品的质量优良,生产成本低廉,具有市场竞争力。