Search

An 11450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptornyces R61 was cloned in Streptornyces lividans using the high-copy-number plasmid pi J702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DDpeptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine β-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coil ampC β-lactamase (class C). Since the Streptomyces R61 DD-peptidase and β-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the β-1actamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the β-lactamases of classes A and C are related in an evolutionary sense. [ABSTRACT FROM AUTHOR]

It was demonstrated that an enzyme system(s) extracted from etiolated seedlings of Stizolobium hassjoo catalyzed the conversion of L-dihydroxyphenylalanine into stizolobinic acid. α-amino-6-carboxy-2-oxo-2H-pyran-3-propionic acid, and stizolobic acid, α-amino-6-earboxy-2-oxo-2H-pyran-4-propionic acid, in the presence of NADP+ or NAD+ under aerobic condition. Enzymically synthesized radioactive stizolobinic acid and stizolobic acid isolated from the reaction mixtures were purified and confirmed to have constant specific radioactivities by cocrystallization with authentic samples. Maximal activity of the enzyme preparation was obtained by using an insoluble polyphenol adsorbent (Polyclar AT) and a reducing agent (araboascorbic acid) in the extraction medium and by subsequent fractionation of the extract with ammonium sulfate followed by Sephadex G-25 gel filtration. Catalytic activity of the enzyme preparation was more unstable under aerobic condition than anaerobic, Attempts to stabilise the enzyme activity were made by the use of many substances which are known to stabilise other enzymes or expected to arrest the inactivation. Evidence is provided in this paper that the previously proposed biosynthetic pathways of stizolobinic acid and stizolobic acid from dihydroxyphenylalanine proceeded in the cell-free system from etiolated seedlings of S. hassjoo. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = √1-q⊃ is fulfilled. In this relation L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load, i.e. the irreversible ATP-utilizing processes in the cell, and q the degree of coupling of oxidative phosphorylation driven by respiration. Since during short time intervals L11 and q are constant whereas L33 fluctuates in the cell, oxidative phosphorylation would only rarely operate at optimal efficiency due to violation of conductance matching. This paper demonstrates that the reversible ATP-utilizing reaction catalyzed by adenylate kinase can effectively compensate deviations from conductance matching in the presence of a fluctuating L33 and hence allows oxidative phosphorylation to operate at optimal efficiency in the cell. Since the adenylate kinase reaction was found to buffer a thermodynamic potential, i.e. the phosphate potential, this finding was generalized to the concept of thermodynamic buffering. The thermodynamic buffering ability of the adenylate kinase reaction was demonstrated by experiments with incubated rat-liver mitochondria. Considerations of changes introduced in the entropy production by the adenylate kinase reaction allowed to establish the theoretical framework for thermodynamic buffering. The ability of thermodynamic buffering to compensate deviations from conductance matching in the presence of fluctuating loads was demonstrated by computer simulations. The possibility of other reversible ATP-utilizing reactions, like the ones catalyzed by creatine kinase and arginine kinase, to contribute to thermodynamic buffering is discussed. Finally, the comparison of the theoretically calculated steady-state cytosolic adenine nucleotide concentrations with experimental data from perfused livers demonstrated that in livers from fed rats conductance matching is fulfilled on a time average and that the degree of coupling corresponded to qpec = 0.97 permitting the most economic maintenance of a maximal output power of oxidative phosphorylation. For the case of livers from starved rats this analysis suggested that the degree of coupling corresponded to qfec = 0.95, permitting the most economic maintenance of a maximal net rate of ATP synthesis at optimal efficiency of oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

By conversion of prochymosin into active chymosin an N-terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology. [ABSTRACT FROM AUTHOR]

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of α-H atoms of the amino acid substrate with 3H2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The α-H of the accumulating j8-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled α-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic β-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of β-H atoms in the amino substrate or of 18O-labelled β-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse α-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonine, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, Ki, is equal to the Michaelis constant, Km, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, α-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic α-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific β-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of α,β-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing β-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). [ABSTRACT FROM AUTHOR]

A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma;-carboxamide groups of protein-bound glutamine residues. These enzymes arc involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a eDNA of guinea-pig liver transglutaminase between the Ncol and Pstl sites of an expression vector, pKK233-2, and produced the Liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyt-terminal sequences. However, the α-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acctylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km, values for substrates in the histamine incorporation into acetyl α1¹-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the Nα-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme. [ABSTRACT FROM AUTHOR]

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide · HCI (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat, values of these chimeras. From the data obtained, two sequences, i.e. 62–103 and 146–220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2-- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other. [ABSTRACT FROM AUTHOR]

Glycogen synthase a was purified over 500-fold by a procedure which involved solubilisation of the enzyme from a protein-glycogen complex by the action of endogenous phosphorylase and debranching enzyme, followed by DEAE-cellulose chromatography, and either gel filtration on Sepharose 4B or fractionation with polyethylene glycol. 15 mg of protein could be obtained from 1000 g of muscle in five days, corresponding to a yield of 20%. The purity was over 90% as judged by gel electrophoresis and ultracentrifugal analysis. The amino acid composition was determined and the absorption coefficient, A1%280nm, measured refractometrically was 13.4. Glycogen synthase a sedimented as two major components, both of which were enzymatically active. The smaller species (13.3S) comprised 85% and the larger species (19.0S) 15% of the material. The molecular weight of the 13.3-S component was determined to be 377000 by high-speed sedimentation equilibrium centrifugation. The subunit molecular weight measured by gel electrophoresis in the presence of sodium dodecylsulphate was 88000 indicating that the 13.3-S species is a tetramer. The properties of the enzyme are compared to those obtained by other workers. [ABSTRACT FROM AUTHOR]

Difficulty may arise in distinguishing between ping-pong and sequential enzyme mechanisms, when using double-reciprocal plots of kinetic data obtained at several fixed values of the concentration of the second substrate; this is because the parallel lines diagnostic of the first case may actually be slowly converging as required by the second. An alternative approach is to choose substrate concentrations so that the second substrate concentration occurs in a number of fixed ratios to the first. A ping-pong mechanism predicts a set of straight lines and a sequential mechanism a set of curved lines (parabolas) in double-reciprocal plots. The method is illustrated by its application to bovinemilk lactose synthetase which requires UDP-galactose and N-acetyl-glucosamine as substrates in a sequential mechanism. [ABSTRACT FROM AUTHOR]

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L- LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G→C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65°C but more stable above 70°C. [ABSTRACT FROM AUTHOR]

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PP1 exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine · ATP complex. Spermidine · ATP is a weaker substrate. The role of spermidine · ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines. [ABSTRACT FROM AUTHOR]

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes 1 and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclcase activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63 000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymenzing activity required Mn2+ but not Mg2+ ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity. [ABSTRACT FROM AUTHOR]

1. A basic protein (pI ≈ 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn- superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25000. This is larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr, 22400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immunoblotting to be exclusively Localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organaspecific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein. [ABSTRACT FROM AUTHOR]

Two 1,3; 1,4-β-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3; 1,4-β-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants. [ABSTRACT FROM AUTHOR]

Cutinase I and cutinase II, two extracellular enzymes produced by Fusarium solani pisi, were shown to be glycoproteins containing 4.3% and 5.1%, carbohydrates, respectively. Upon treatment with alkali both enzymes generated chromophores which absorbed at 241 nm. Treatment of both proteins with alkaline NaB3H4 gave labeled protein and labeled monosaccharides. Hydrolysis of the labeled protein followed by chromatographic and enzymatic analyses of the products showed that alanine, 2-aminobutyrate, phenylalanine, tyrosine and L-gulonic acid accounted for nearly all of the 3H contained in the protein. The four labeled amino acids were shown to be 1:1 mixture of D and L isomers and 3H was nearly equally distributed between α and β positions in each amino acid. The N-terminal amino group of cutinase I did not react with either phenylisothiocyanate or dansyl chloride. This amino group was suggested to be in amide linkage with glucuronic acid because upon treatment of the protein with neutral NaB3H4, gulonic acid attached to the protein became labeled and only gulonic acid was labeled when the protein was deglycosylated with HF prior to alkaline NaB3H3 treatment. Furthermore, N-gulonylglycine was isolated from the pronase digest of the labeled protein. Chromatographic identification and quantification of the labeled carbohydrates released from cutinase I by alkaline NaB3H4 showed that one mole of cutinase I has one mole each of mannose, arabinose, N-acetylglucosamine, and glucuronic acid O-glycosidically linked to serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine. In addition, the N-terminal glycine is in amide linkage with glucuronic acid. Since almost identical experimental results were obtained with cutinase II this protein is also suggested to have the same structural features as those suggested above for cutinase I. [ABSTRACT FROM AUTHOR]

The arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-l-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent from digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation of the peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains. [ABSTRACT FROM AUTHOR]

The effects in vitro of D-amino acids or glycine on the formation of wall-bound peptidoglycan were studied with wall membrane enzyme preparations from Gaffkya hornari. These amino acids inhibited the incorporation of nascent peptidoglycan into the preformed polymer (e.g. ID50 values for D-alanine, D-leucine, and glycine = 5.6 mmol/l, 1.3 mmol/l, and 11 mmol/l, respectively). The inhibition was accompanied by an incorporation of the inhibitor into position 4 of the peptide subunit Ala1-DGlu2(Lys3-DAla4), where the indices refer to the position of an amino acid residue within the peptide subunit. It is suggested that the reaction is catalyzed by an LD-carboxypeptidase. Therefore, this enzyme has also D-amino acid exchange activity. At inhibitory concentration fewer tripeptide subunits were formed in the nascent peptidoglycan in favour of the formation of tetrapeptide subunits bearing the inhibitor at the C termini. The tripeptide subunits are assumed to be necessary in order that nascent peptidoglycan is utilized as substrate in the transpeptidation reaction. Thus an essential role of the LD-carboxypeptidase is indicated. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions. [ABSTRACT FROM AUTHOR]

1. The cyanobacterium Aphanocapsa 6714 which grows in the dark on D-glucose, will take up Dglucose and the analogue 3-O-methyl-D-glucose; uptake of each of these compounds was inhibited competitively by the other and by 6-deoxy-D-glucose. 2. This cyanobacterium accumulated 3-O-methyl-D-glucose up to 100-fold relative to the medium but did not modify or metabolize it to a significant degree. 3. Intracellular 3-O-methyl-D-glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D-glucose and 3-O-methyl-D-glucose. 4. Although not characterized to the same extent, D-glucose and 3-O-methyl-D-glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D-glucose, was similar. 5. Other cyanobacteria that do not grow on D-glucose take up this compound at much lower rates which were unaffected by analogues of D-glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac. 6. It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D-glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D-glucose as a growth substrate. [ABSTRACT FROM AUTHOR]

1. The reaction of native chorismate mutase/prephenate dehydratase with 5,5′-dithio-bis(2nitrobenzoic acid) (Nbs2) caused a differential inactivation of the two enzymic activities. The dehydratase activity was completely inactivated by the reaction of 1.2 mol of sulphydryl per mol of enzyme subunit, while the mutase activity was inhibited only 5%. The Michaelis constant for chorismate increased about 2.5-fold while the maximum rate for the mutase activity was unaffected. Reaction of an average of two more moles of sulphydryl per mole of enzyme subunit caused a further 25% inhibition of the mutase activity. 2. Circular dichroic measurements indicated that native chorismate mutase/prephenate dehydratase has 25% α-helix and that modification of one sulphydryl group per subunit with Nbs2 caused significant changes in the secondary structure of the enzyme and in the environment of one or more tyrosine residues. 3. Prephenate is a competitive inhibitor of the mutase activity, with an inhibition constant of 0.047 ± 0.008 mM. Modification of one sulphydryl group per subunit with Nbs2 increased the value of the inhibition constant and the Michaelis constant for chorismate by the same amount. 4. At concentrations from 5-1000 µM the allosteric inhibitor phenylalanine did not markedly affect the reactivity of the most reactive sulphydryl group with Nbs2, but decreased the reactivity of the other sulphydryl groups. 5. Prephenate protected the most reactive sulphydryl group against reaction with Nbs2; phenylpyruvate had no effect. 6. It was concluded that one sulphydryl group is involved directly in the dehydratase active site. 7. The results are interpreted as favouring the theory that chorismate mutase/prephenate dehydratase has separate active sites for the two activities, although the possibility of partially overlapping sites has not been eliminated. [ABSTRACT FROM AUTHOR]

2-Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (εmax = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives. [ABSTRACT FROM AUTHOR]

Ribonuclease L (RNase L) is a key enzyme in the 2-5A host defense system, and its activity is strictly regulated by an unusual 2′,5′-linked oligoadenylate (2-5A). A bipartite model, in which the N-terminal half of RNase L is responsible for the 2-5A binding and the C-terminal half alone is able to hydrolyse the substrate RNA, has been proposed on the basis of the results of deletion mutant analyses [Dong, B. & Silverman, R.H. (1997) J. Biol. Chem. 272, 22236–22242]. Above all, the region between Glu711 and His720 was revealed to be essential for RNA binding and/or hydrolysis. To dissect the function of the region, we performed scanning mutagenesis over the 10 residues of glutathione S-transferase (GST)-fusion RNase L. Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding acitivity. The losses of the RNase activity were not restored by its conservative mutation, whereas the RNA binding activity was enhanced in the case of Y712F. These results indicate that both Tyr712 and Phe716 provide the enzyme with a RNA binding activity and catalytic environment. [ABSTRACT FROM AUTHOR]

Information on the regulation and structure–function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer–tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern. [ABSTRACT FROM AUTHOR]

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN [Moreau et al. (2002). Arch. Virol. 147, 1761–1778]. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein–protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/ΔC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends. [ABSTRACT FROM AUTHOR]

The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed. [ABSTRACT FROM AUTHOR]

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group. [ABSTRACT FROM AUTHOR]

Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56 000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168 000). Treatment with dimethylimidates of other chain length yields significantly, less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with monofunctional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains. [ABSTRACT FROM AUTHOR]

Human adenylate kinase (isoenzyme AK11) from skeletal muscle is a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine at the C-terminus. The primary structure of the enzyme was determined : [This symbol cannot be presented in ASCII format] When the primary structure of the human enzyme was fitted to the electron density map of porcine adenylate kinase, all nine amino acids which are different in the homologous enzymes from pig and man were located on the surface of the molecule. Precession photographs of crystalline human and of crystalline porcine adenylate kinase corroborated the result that the polypeptide chains of the two enzymes are folded in a closely related rnanner. The structure of human adenylate kinase incorporates the so-called nucleotide-binding domain which is present in a wide variety of proteins in nature. Some implications of this phenomenon for the molecular biology and the molecular pharmacology of man are discussed. [ABSTRACT FROM AUTHOR]

Ribonuclease A has been guanidinated at the lysine residues and the nona-guanidinated and deca-guanidinated (fully substituted) products separated. In confirmation of an earlier report by Glick and Barnard (1970), it has been shown by chemical procedures that the former derivative is not reacted at lysine-41. Guanidination of lysine-41 to produce the fully substituted product causes loss of enzymic activity without any apparent change of conformation, as tested by conformational comparisons (using proton magnetic resonance spectroscopy) including (a) difference spectroscopy. (b) heat denaturation and (c) titration of the active-site histidine residues 12 and 119. This is good evidence for the involvement of lysine-41 in a catalytic role in the enzyme. Dimethylation of lysine-41 of nona-guanidinated ribonuclease A produces sharp proton resonances which shift as the dimethylamino group is titrated and allow the determination of an apparent pK of 8.8 for ansubstituted lysine-41. [ABSTRACT FROM AUTHOR]

The binding of 10 isomeric α-amino-heptanoic acids, of two isomeric α-amino-octanoic acids, of isoleucinol and valinol, and of various α-hydroxy acids to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not-too-small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three-point-attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the α-hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety. Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the/3-carbon atom. The functioning of the side-chain recognition site is best understood by imagining a two-finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cβ epimer. [ABSTRACT FROM AUTHOR]

Using 14C-labeled Salmonella bacterial cells as the substrate, the enzymic and molecular properties of the base-plate parts of phage P22 were studied. The base-plate part consisted of a single protein species which cleaved extensively the O-antigen of Salmonella typhimurium, Salmonelly schottmuellerie and with somewhat slower rate that of Salmonella typhi, releasing oligosaccharide products with rhamnose at the reducing end. Much less cleavage was observed with a strain of S. typhimurium lysogenie for P22, and no significant reaction with Salmonella anatum. Salmonella newington and Salmonella minneapolis. The base-plate part enzyme was a very heat-stable protein and only 10–20% loss was observed after treatment at 85 °C for 5 min. The pH optimum of the enzyme was around 7.5. and the glycosidase activity was not influenced by the ionic strength (25–250 mM) of the medium or the presence of Mg2+. The molecular weight of the base-plate part was 320000 by sedimentation equilibrium. Dodecylsulphate-acrylamide gel electrophoresis revealed a single band of molecular weight 77000, indicating that a single base-plate part corresponds to a tetramer of identical subunits. Circular dichroism spectra of P22 base-plate parts showed a major contribution of β structure. The protein was rich in acidic amino acids, glycine and serine. [ABSTRACT FROM AUTHOR]

Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. The crystalline alcohol oxidases of both yeasts oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to for-mate, and were inactivated by relatively low concentrations of hydrogen peroxide. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face. [ABSTRACT FROM AUTHOR]

1. D-Gtyceraldehyde-3-phosphate dehydrogenase fi'om an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized. 2. Tile enzyme was found to possess remarkable heat stability, being slowly inactivated at 90°C. 3. Basic kinetic constants and pH profile are reported. The enzyme was activated 25-fold by 90 mM NH4Cl, and also by ethanol up to 5-fold at 30°C. 4. The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme. 5. The enzyme was shown to be a tetramer of molecular weight 130000–135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme. 6. The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources. [ABSTRACT FROM AUTHOR]

A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside tripbosphates) to be detected. This modification consists in a previous fixation of DNA · enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3–0.5% and with fixation time between 2 rain and 100 rain. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCI) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400–500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20°C. At temperatures above 30°C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20&Deg;C and 30°C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde. [ABSTRACT FROM AUTHOR]

L-Isoleucine: tRNA ligase from Escherichia coli could be irreversibly inactivated by L-isoleucyl-bromomethyl ketone but not by L-isoleucyl-chloromethyl ketone. The inactivation rate exhibited a saturation concentration dependence typical for an affinity reagent. L-Isoleucine provided 100% protection against inactivation at saturating concentration, whereas ATP, AMP, and pyrophosphate offered partial protection and tRNAIle, no protection. The ligase was labelled in preparative scale with L-[14C]isoleucyl-bromomethyl ketone. The molar ratio of label incorporated to enzyme inactivated was close to unity. The protein was subsequently subjected to tryptic digestion and the radioactive peptide isolated and identified. The labelled amino acid proved to be the same cysteine previously reported as being labelled with N-[14C]ethylmaleimide [Kula, M.-R. (1974) FEBS Lett. 46, 130–133]. [ABSTRACT FROM AUTHOR]

Ornithine carbamoyltmnsferase from £scherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coil W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme, Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofoloate (26.1-27.0 U/mg) and folate (1.3 - 2.2 U/mg), and had identical molecular weights (23 5000) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity. [ABSTRACT FROM AUTHOR]

1. The symmetry of proteins composed of identical polypeptide chains has been investigated by means of cross-linking with bifunctional reagents and subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis. The majority of the investigations were performed with diimidates of different chain lengths (C3 - C12), which react exclusively with amino groups. Aldolase, catalase, fumarase, pyruvate kinase, tetrameric proteins with identical polypeptide chains, reveal a D2 symmetry, i.e. they appear to be composed of two pairs of polypeptide chains. The validity of this conclusion is demonstrated with lactate dehydrogenase. This enzyme, shown by X-ray analysis to have a D2 symmetry, yields after cross-linking and subsequent polyacrylamide electrophoresis the band pattern expected for a protein with this quaternary structure and similar to the pattern obtained with the above enzymes. 2. The influence of the experimental conditions on the cross-linking reaction has been investigated. The selectivity of the bifunctional reagent for the different contact domains within the quaternary structure of a protein depends on the reaction time, the chain length and on the concentration of the reagent. In general the D2 symmetry becomes more obvious with increasing chain length and with increasing concentration of the diiniiidate. Diethylpyrocarbonate showed very little selectivity. 3. Problems and limitations of the method have been investigated. The four polypeptide chains of β-galactosidase could not be cross-linked with the investigated reagents. Glyceraldehyde-phosphate dehydrogenase barely showed the predicted cross-linking pattern, although its D2 symmetry has been demonstrated by X-ray analysis. Yeast alcohol dehydrogenase shows a pattern which did not indicate any substructure with respect to the quaternary structure. Catalase yielded a complicated band pattern indicating that the cross-linking prevented complete unfolding of the polypeptide chains in sodium dodecylsulfate. These pattern did not occur after cross-linking with diethyl pyrocarbonate. 4. The applicability of molecular-weight determinations with sodium dodecylsulfate - polyacrylamide gel electrophoresis to cross-linked proteins has been investigated. Calibrations obtained with cross-linked proteins are very similar to that obtained with non-cross-linked polypeptide chains. Even with catalase the RF values of the predominant bands fit into this calibration. [ABSTRACT FROM AUTHOR]

The topography of the active site of histidyl-tRNA synthetase has been investigated by determining Ki values for a variety of structural analogues of histidine, using the ATP-PPi exchange and tRNA aminoacylation reactions. Using these kinetic constants it has been possible to have a measure of the relative binding affinity of the enzyme for the histidine analogues. The following conclusions have been drawn: (a) the enzyme is stereospecific in the formation of aminoacyl-tRNA complexes, since the D-isomer of histidine does not influence the two reactions; (b) the carboxyl group is not required for binding; (c) bulky derivatives of the carboxyl group prevent the molecules from binding to the enzyme; (d) the amino group permits a good binding affinity; (e) the length of the ring side chain plays a very important role as point of attachment to the enzyme; (f) the kinds of-heteroatoms on the ring determine the inhibitory properties of the analogues. [ABSTRACT FROM AUTHOR]

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the β chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified eysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide. [ABSTRACT FROM AUTHOR]

Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, α of molecular weight 73000 and β of molecular weight 63000. The enzyme is an asymmetric tetramer α2β2, which binds two moles of each ligand per mole. Incubation of the purified enzyme with trypsin results in an irreversible conversion: the α-subunit remains apparently unchanged but β is rapidly degraded and yields a lighter species β′ of molecular weight 41000. The trypsin-modified enzyme is an α2β′2 molecule which can still activate phenylalanine but cannot transfer it to tRNAPhe furthermore it does not bind tRNAPhe but its kinetic parameters are identical to those of the native enzyme width respect to ATP and phenylalanine. Therefore the two β subunits play a critical part in tRNA binding. Isolated α or β′ subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation. [ABSTRACT FROM AUTHOR]

The interaction of L-tyrosine, L-tyrosyladenylate and tRNATyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 × 44000), binds only a single molecule of L-tyrosine per dimer with a Kd of 2 × 10-5 M at pH 7.8 and 23 °C. The tyrosyl-tRNA synthetase-tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNATyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having Kd values of 2 × 10-7 M and 1.3 × 10-6 M respectively at pH 6.5 and 25 °C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme. [ABSTRACT FROM AUTHOR]

Immunochemical techniques were used to study the mechanism underlying the marked increase in the level of acetyl-coenzyme A carboxylase activity in chick liver observed after hatching. The results of immunochemical titrations and Ouchterlony double-diffusion analysis indicated that this increase in the activity level of the enzyme was due to an elevation in the enzyme quantity. Isotopic leucine incorporation studies revealed that the rate of synthesis of the enzyme per liver was 18-fold higher in 9-day-old chicks than in 1-day-old chicks. In terms of the synthesis rate per gram of liver, this increase was 5-fold. The half-life for degradation of the enzyme in 9-day-old chicks was shown to be 46 h, whereas no apparent degradation of the enzyme as well as of total soluble liver protein was observed in 1-day-old chicks. These results indicate that the increase in the hepatic acetyl-CoA carboxylase content in growing chicks can be ascribed to accelerated synthesis of the enzyme. [ABSTRACT FROM AUTHOR]

The location of amino acid residues within the tobacco mosaic virus protein subunit is discussed. Sequence data, X-ray crystallographic measurements, and the availability of specific residues for enzymic, immunological or chemical reaction are amongst the information used to trace roughly how the tobacco mosaic virus polypeptide chain winds in and out from the virus axis. Published rules for predicting secondary structure are then applied to obtain a diagram of the course of the polypeptide chain. This map should be useful for the interpretation of X-ray diffraction data and already permits an outline of the main features of the inner third of subunit to be suggested. [ABSTRACT FROM AUTHOR]