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The enhancement of the catalytic power of enzymes is a subject of enormous interest both for science and for industry. The latter, in particular, due to the vast applications enzymes can have in industrial processes, for instance in the desulfurization of crude oil, which is mandatory by law in many developed countries and is currently performed using costly chemical processes. In this work we sought to enhance the turnover rate of DszD from Rhodococcus erythropolis, a NADH-FMN oxidoreductase responsible for supplying FMNH2 to DszA and DszC in the biodesulfurization process of crude oil, the 4S pathway. For this purpose, we replaced the wild type spectator residue of the rate limiting step of the reduction of FMN to FMNH2, a process catalysed by DszD and known to play an important role in the reaction energy profile. As replacements, we used all the naturally occurring amino acids, one at a time, using computational methodologies, and repeated the above-mentioned reaction with each mutant. To calculate the different free energy profiles, one for each mutated model, we applied quantum mechanics/molecular mechanics (QM/MM) methods within an ONIOM scheme. The free energy barriers obtained varied between 15.1 and 29.9 kcal mol−1. Multiple factors contributed to the different Δ G values. The most relevant were electrostatic interactions and the induction of a favourable alignment between substrate and cofactor. These results confirm the great potential that chirurgic mutations have for increasing the catalytic power of DszD in relation to the wild type ( wt) enzyme. [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]

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]

Serine acetyltransferase (SAT), is an enzyme that has been shown to catalyze the conversion of acetyl-CoA and L-serine to CoA and O-acetyl- L-serine in microorganisms and higher plants. SAT is a member of a family of hexapeptide- containing transferases. All dimer-of-trimer interactions are mediated through amino acids within an N-terminal extension common only to a subset of SAT, suggesting that members of this subfamily may also adopt hexameric structures. Comparisons of the Haemophilus influenzae SAT structure with related enzymes suggest by analogy that the active sites in Haemophilus influenzae SAT are likely to be formed by a crevice between adjacent threefold-related protomers.

Regulation of specific protein function is of great importance for both research and therapeutic development. Many small or large molecules have been developed to control specific protein function, but there is a lack of a universal approach to regulate the function of any given protein. We report a general host–guest molecular recognition approach involving modification of the protein functional surfaces with genetically encoded unnatural amino acids bearing guest side chains that can be specifically recognized by cucurbit[7]uril. Using two enzymes and a cytokine as models, we showed that the activity of proteins bearing unnatural amino acid could be turned off by host molecule binding, which blocked its functional binding surface. Protein activity can be switched back by treatment with a competitive guest molecule. Our approach provides a general tool for reversibly regulating protein function through molecular recognition and can be expected to be valuable for studying protein functions. [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]

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]

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]

Summary: The aim of this study was to develop a green ultrasound‐assisted enzymatic process to separate protein from brewer's spent grain (BSG) to produce protein hydrolysates and determine the physicochemical properties of produced protein hydrolysates. When the enzyme (Alcalase) loading increased from 1 to 20 μL g−1 BSG, the protein separation efficiency increased from 34.0% to 61.6%. The application of ultrasound pretreatment further increased protein separation efficiency to 69.8%. More promisingly, the ultrasound pretreatment was able to reduce enzyme loading by 73% and decrease enzyme incubation time by 56%. The produced protein hydrolysates had molecular weights lower than 15 kDa and high protein solubilities at the pH of 1.0–11.0. The ultrasound pretreatment improved the protein solubility to above 90%. Glutamic acid and proline were the most abundant amino acids in produced protein hydrolysates. This study demonstrated that enzymatic hydrolysis along with ultrasound pretreatment is an effective way to separate protein from BSG. [ABSTRACT FROM AUTHOR]

Objective: Heightened somatic symptoms are reported by a wide range of patients with chronic pain and have been associated with emotional distress and physical dysfunction. Despite their clinical significance, molecular mechanisms leading to their manifestation are not understood.Methods: We used an association study design based on a curated list of 3,295 single nucleotide polymorphisms mapped to 358 genes to test somatic symptoms reporting using the Pennebaker Inventory of Limbic Languidness questionnaire from a case-control cohort of orofacial pain (n = 1,607). A replication meta-analysis of 3 independent cohorts (n = 3,189) was followed by functional validation, including in silico molecular dynamics, in vitro enzyme assays, and measures of serotonin (5-HT) plasma concentration.Results: An association with the T allele of rs11575542 coding for an arginine to glutamine substitution in the L-aromatic amino acid decarboxylase (AADC) enzyme was replicated in a meta-analysis of 3 independent cohorts. In a combined meta-analysis of all cohorts, this association reached p = 6.43 × 10-8 . In silico studies demonstrated that this substitution dramatically reduces the conformational dynamics of AADC, potentially lowering its binding capacity to a cofactor. in vitro enzymatic assays showed that this substitution reduces the maximum kinetic velocity of AADC, hence lowering 5-HT levels. Finally, plasma samples from 90 subjects showed correlation between low 5-HT levels and heightened somatic symptoms.Interpretation: Using functional genomics approaches, we identified a polymorphism in the AADC enzyme that contributes to somatic symptoms through reduced levels of 5-HT. Our findings suggest a molecular mechanism underlying the pathophysiology of somatic symptoms and opens up new treatment options targeting the serotonergic system. ANN NEUROL 2019;86:168-180. [ABSTRACT FROM AUTHOR]

Exerting control over the size, morphology, and complexity of metal and semiconductor nanoparticles and nanostructures is a requisite for exploring novel phenomena, and the potential applications of these nanomaterials. Bottom-up colloidal chemistry syntheses can benefit from using biomolecules as active elements to influence the formation of inorganic nanoparticles. In this review, we will discuss how three main biomolecule types, (namely DNA; amino acids, peptides, and proteins; and enzymes), can affect the growth of metal and semiconductor nanoparticles. We will present and discuss the templating and non-templating roles of those biomolecules, featuring key aspects and prospects of biomolecule-assisted metal and semiconductor nanoparticle growth. [ABSTRACT FROM AUTHOR]

Although the formation mechanism of biogenically metallic nanoparticles is broadly associated to enzyme mediation, major attention has been given to the role of proteins and peptides in oxido‐reduction of metallic ions leading to these nanostructures. Among the wide range of biomolecules that can act not only as capping agents but also as non‐enzymatic agents to form nanoparticles, disulphide bridge‐containing peptides and amino acids particularly stand out. The literature proposes that they actively participate in the process of nanoparticles' synthesis, with thiols groups and disulphide bridge moieties as the reaction catalytic sites. Similarly, denaturated enzymes containing exposed S–S or S–H moieties are also able to reduce metallic ions to form nanoparticles. This mini‐review is focused on the biogenic synthesis of metallic nanoparticles such as gold, silver, copper, platinum, palladium, lead and selenium, in which proteins, peptides, reductases and even oxido‐reductases act as non‐enzymatic catalysts of the reduction reaction, opening economically and ecologically favourable perspectives in the nanoparticles synthesis field. [ABSTRACT FROM AUTHOR]

Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes containing foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones. [ABSTRACT FROM AUTHOR]

The article discusses the need for tight control of nutrient-induced signaling related to precise regulation of intracellular trafficking in nutrient transporters. It indicates that there are numerous examples of transporters more expressed in the absence of their substrate in addition to extracellular nutrients that can trigger induction of transporters and enzymes. It suggests that Gap1 amino acid transceptor can be seen more in view of its signaling function than its transport function.

A morpheein is a homo-oligomeric protein that can exist as an ensemble of physiologically significant and functionally distinct alternate quaternary assemblies. Morpheeins exist in nature and use conformational equilibria between different tertiary structures to form distinct oligomers as a means of regulating their function. Notably, alternate morpheein forms are not misfolded forms of a protein; they are differently assembled native states that contain alternate subunit conformations. Transitions between alternate morpheein assemblies involve oligomer dissociation, conformational change in the dissociated state, and reassembly to a different oligomer. These transitions occur in response to the protein's environment, for example, effector molecules, and represent a new model of allosteric regulation. The unique features of morpheeins are being revealed through detailed characterization of the prototype enzyme, porphobilinogen synthase, which exists in a dynamic equilibrium of a high activity octamer, a low activity hexamer, and two dimer conformations. Morpheeins are likely far more common than previously appreciated. There are, however, both intellectual and experimental barriers to recognizing proteins as morpheeins. These barriers derive from the way we were taught and continue to teach about protein folding, protein purification, protein structure-function relationships, and enzyme kinetics. This article explores some of these limitations and encourages incorporation of morpheeins into both introductory and advanced biochemistry classes. [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]

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Conditional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factorstimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 β (GSK3β) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3β phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3β signalling. Consistent with this, active, but not H154D mutant or the related PP2Cα, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3β phosphorylation. [ABSTRACT FROM AUTHOR]

The presence of endoproteinases in the intercellular washing fluid of the first wheat (Triticum aestivum) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously submitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases. [ABSTRACT FROM AUTHOR]

Changes in the amounts of enzyme proteins and mRNAs for phenylalanine ammonialyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 6.-.-.-) were investigated in suspension cultures of carrot (Daucus carota L. cv. Kurodagosun), in which anthocyanin synthesis was induced and repressed in the absence and presence of 2,4-dichrolophenoxyacetic acid (2,4-D). respectively. The amounts of enzyme proteins were measured using anti-PAL and anti-CHS sera. In a medium lacking 2,4-D, enzyme proteins and mRNAs for both PAL and CHS were induced 5 days after transfer, when the anthocyanin synthesis was induced. They all increased coordinately during days 5-7 after transfer, when anthocyanin was rapidly synthesized, and then they decreased. In contrast, in a medium containing 2,4-D, the amount of enzyme protein and mRNA for CHS were below detectable levels throughout the culture time. As regards PAL, however, whether 2,4-D was present or not, both enzyme protein and mRNA rapidly increased after the transfer of cells to fresh medium, and then decreased and remained at low levels in a medium containing 2,4-D. The addition of 2,4-D to cells cultured for 6 days in a medium lacking 2,4-D caused an immediate decrease in the amounts of enzyme proteins and mRNAs for both PAL and CHS. All the results obtained here indicate that the induction and suppression of the synthesis of PAL and CHS were probably due to increase and decrease, respectively, in the amounts of their mRNAs. Thus, it is suggested that the induction and suppression of anthocyanin in this system may be regulated at the transcriptional level by 2,4-D. [ABSTRACT FROM AUTHOR]

Plants of Urtica dioica L., a very nitrophilous species, were grown in a nutrient solution containing either high (100%) or low (2%) nutrient supply. Part of these plants were subjected to a sudden switch from 100% to 2% or vice versa. Plant weight, sugar and organic nitrogen (both soluble and insoluble) and nitrate content were measured during growth. The activities of two nitrogen assimilating enzymes, nitrate reductase (NR) and glutamine synthetase (GS) were determined. Growth of Urtica dioica was retarded at low nutrient supply. Root growth was limited by another factor than nitrogen. This was shown by a higher protein content in the first period after a switch from 100% to 2%, redistribution of nitrogen from shoot to root could be demonstrated, and leakage from the root into the nutrient solution. It is suggested that in these conditions GS in the root reacted to this downward flux. Comparison with earlier findings on the less nitrophilous Plantago lanceolata showed that at 100% nutrient supply a correlation occurs between nitrate reduction and glutamine synthetase activity in that plant part which exported reduced nitrogen: the root in P. lanceolata and the shoot in U. dioica. In the importing plant part, glutamine synthetase was influenced by nitrate reduction as well as by imported reduced nitrogen. [ABSTRACT FROM AUTHOR]

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD+-glutamate dehydrogenase (oxidative de-amination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa (Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N2-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD+-glutamate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of a folic acid synthesis (fas) gene from Pneumocystis carinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an Mr of 83979. A recombinant baculovirus was constructed which directed expression of the predicted Fas740 polypeptide in cultured Spodoptera frugiperda (SF9) insect cells. The overexpressed ‘full-length’ protein migrated anomalously in sodium dodecyl sulfate/polycrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69-kDa species was also recognized by polyclonal sera specific for the Fas protein in immunoblotting analyses. Dihydroneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroate synthase activities were readily detected in SF9 extracts in which the 71.5/69-kDa immunoreactive species were overproduced, demonstrating that three enzyme functions involved in catalyzing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in P. carinii. Importantly, the polyclonal sera recognize a single polypeptide, although the nature of the suggested post&shyptranslational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon amino acid sequence similarly to their bacterial counterparts is discussed. Furthermore, expression of various truncated fas gene constructs demonstrates that the complete fas ORF, including the N-terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydroteroate synthase (FasD domain) activites. This suggests interactions between the domains within the larger polypeptide to stabilized the functions of these two enzymes. The FasC domain, which contains 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity, is able to fold and function independently of the other domains. The requirement by mammalian cells for preformed folates, and the absence of dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydropteroate synthase from these tissues opens up the possibility of designing higly selective drugs which inhibit these unique targets. [ABSTRACT FROM AUTHOR]

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation- exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 k Da by SDS/PAG E. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 750 of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half-life at 70°C was 2.85 rain. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the PI position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites. [ABSTRACT FROM AUTHOR]

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-1,7-bisphosphatase [Sed(1,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72–507 bp) and seven introns (85–626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-1,6-bisphosphatase (Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(1,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(1,7)P2ase enzyme. [ABSTRACT FROM AUTHOR]

Sorbin has been isolated from extracts of porcine upper intestine, and the biological activity in absorbing water and electrolytes utilized to monitor the purification prozedure. Pure sorbin was obtained in a yield of about 1 mg/Mg boiled intestine. The protein chain has 153 amino acid residues and the primary structure was determined by analyses of CNBr-cleaved fragments and four enzymatic digests. The protein has a free N-terminal Met and an amidated C-terminal Ala. No structural similarity was observed with other known proteins in data bases, but several segments have special properties and the C-terminal half is rich in Pro and Arg. [ABSTRACT FROM AUTHOR]

A cDNa clone for the subunit Vc, the smallest nuclear-encoded subunit, of sweet potato cytochrome c oxidase was isolated. Nucleotide sequence analysis showed that the subunit is synthesized as a precursor from which only the amino-acid, methionine, residue is removed to form the mature subunit. The mature subunit consists of 63 amino acid residues which are arranged so as to form one hydrophobic cluster lying between two hydrophilic clusters; such an arrangement is also seen with some nuclear-encoded small subunits of the enzymes from other eukaryotes. The labeled precursor protein was synthesized with the cDNA by in vitro transcription (SPs system) and translation (wheat germ system). This precursor was imported into isolated sweet potato tuberous root mitochondria without any significant processing, the import being dependent on membrane potential. [ABSTRACT FROM AUTHOR]

The S′-subsite specificity of bovine pancreatic α-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S′ binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude. [ABSTRACT FROM AUTHOR]

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1. [ABSTRACT FROM AUTHOR]

1. Diacyiglycerol kinase apoprotein was purified from membranes of Escherichia coli K 12 by a six-step procedure that included HPLC. The proposed assignment of the enzyme to the dgkA gene [Lightner et a!. (1983) J. Blot Chem. 258, 10856-10861] could be supported by molecular mass determination (≈ 14 kDa), N-terminal sequencing (Met-Ala-Aso), cyanogen bromide fragmentation and amino acid analysis. As predicted, proline was absent. 2. The membrane-associated as well as the butan-1-ol-dissolved enzyme survived heating to 100°C. 3. Alkylglycoside detergents were found to constitute an additional class of lipid activators. 4. The enzyme apoprotein in a non-activating substrate/detergent solution was capable of autocatalytic self-activation which was attributed to a novel feedback activation mechanism involving phosphatidic acid (diacylglycerol 3-phosphate). [ABSTRACT FROM AUTHOR]

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1°C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 × 107 M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+ FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+ which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-it-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP ⇄ MgADP + ADP) from baker's yeast has been determined. 1. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid-phase sequencer or a gas-phase sequencer. 2. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys-C and cyanogen bromide cleavages. 3. The N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. 4. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages. 5. The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active-site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP : AMP phosphotransferase from beef heart mitochondria. [ABSTRACT FROM AUTHOR]

Assignments in the ¹H NMR spectrum for more than 120 resonances arising from 38 of the 130 amino acid residues of human lysozyme are presented. Assignments have been achieved using a combination of one and two-dimensional NMR techniques. Two-dimensional double-quantum correlated spectroscopy and relayed coherence transfer spectroscopy were found to be particularly useful for the identification of spin systems in the aromatic and methyl regions of the spectrum. These spin systems were assigned to specific residues in human lysozyme with reference to the X-ray crystal structure using one-dimensional nuclear Overhauser enhancement (NOE) data and a computer-based search procedure. Unique assignments were found for resonances of 27 amino acid residues even when a distance constraint on NOE effects of 0.7 nm was used in the search procedure; for the remaining residues closer constraints or additional informational were required. The assignments include all but one of the resonances in the aromatic region of the spectrum and all the methyl group resonances in the region upfield of 0.6 ppm. The assignments presented here provide a basis for a comparison of the NMR spectra of human lysozyme and the more widely studied hen lysozyme. [ABSTRACT FROM AUTHOR]

A carboxylesterase {2,3,4,6-tetra-O-acetyl-l-[(N-acetyl-N-phenylamino)oxy]-1-deoxy-β-D-glucopyranoside (GPA) O-deacetylase} from a culture product of Aspergillus oryzae (Taka diastase) was purified 8500-fold with a yield of 3 %. The molecular mass of the purified enzyme was shown to be 35 ± 1 kDa by SDS/PAGE. The enzyme shows a selective O-deacetylation activity of GPA to give the fully O- deacetylated glucoside. Among the substrates tested, the enzyme did not hydrolyze benzoyl and phenyl-acetyl esters and acetamides. In the hydrolysis of p-nitrophenyl esters, the acyl preference is acetyl > propionyl > butyryl, judging from the VmaxIKm values. A good correlation between log(VmaxIKm) and the Taft's Esconstant of the alkyl group of the acyl moiety was obtained. The optimum pH was around 7.3 at 37°C, and the enzyme was inhibited by mercuric chloride, p-chloromercuribenzoate and diisopropyl fluorophosphate. This enzyme should be useful for the selective removal of acetyl groups that serve to protect hydroxyl groups during carbohydrate synthesis. [ABSTRACT FROM AUTHOR]

The heterogeneity of the human amyloid proteins SAA and AA was studied. Both proteins could be separated into several fractions by ion-exchange chromatography. Amino acid analysis of the ion-exchange-chromatographed fractions of protein AA showed that the main difference was in the length of the polypeptide. Thus. it seems that the original AA preparation consists of a mixture of AA proteins with length ranging from 66 to 78 amino acid residues. By enzymatic degradation of three different forms of SAA with kallikrein, fragments were formed with a molecular weight very similar to that of protein AA. [ABSTRACT FROM AUTHOR]

Threonine dehydratase is the key enzyme in L-isoleucine synthesis, since it is allosterically feedback-inhibited by L-isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val-323-Ala, which is characterized by the fact that the L-isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high-activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild-type enzyme. Another peculiar mutant generated is the double mutant His-278-Arg-Leu-351-Ser. Here, again, L-isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L-isoleucine-dependent loss of pyridoxal-5'-phosphate from this mutant enzyme. Further enzymes obtained are reduced in L-isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical β-subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of threonine dehydratase. [ABSTRACT FROM AUTHOR]

The tripeptide Nα,N∊-diacetyl-lysyl-D-alanyl-D-alanine (Ac2-LLys1-DAla2-DAla3), which is the standard substrate of the Zn2+G and serine R61 D-alanyl-walanine peptidases, and several LDD tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short-range and long-range interactions. Among the many backbone conformers of minimal energy of the &phis;i,ψi space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90 % or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ΣP values arc still as high as 35-50 %. All the above tripeptides bind to the serine D-alanyl-D-alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+ D-alanyl-D-alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1. 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2-LLys-DAla-DAla show that the main backbone has a tendency to adopt a ring-like shape from which the LLys side chain protrudes as an extended structure. This latter structure forms with the C-terminal D-alanyl-D-alanine an angle varying between 120° and 180° (depending on the conformers) so that its N-terminal acetyl group is about 1 -1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a DAla at position 2 with both D-alanyl-D-alanine peptidases and at position 3 in the case of the serine D-alanyl-D-alanine peptidase. Finally, all the conformers of the LAla2 and LA1a3 analogues of Ac2-LLys-DAla-DAla fall outside the backbone conformational space that comprises the &phis;i&phe;i angles exhibited by the four types of conformers of the LDD tripeptides. The LAla2 and LAla3 tripeptide analogues do not bind to the serine D-alanyl-D-alanine peptidase (at least at a 10 mM con- centration) but they behave as noncompetitive inhibitors of the Zn2+ D-alanyl-D-alanine peptidase. [ABSTRACT FROM AUTHOR]

The major polypeptide chain of bovine β-crystallin, βBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the βBp chain. The N-terminal alanine residue was shown to be N-α-acetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23 210. There is a considerable degree of homology between the N-terminal and C-terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of βBp is sufficiently related to that of γ-crystallin II to place these proteins in the same superfamily. No sequence relationship was found with the α-crystallin chains. [ABSTRACT FROM AUTHOR]

The tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus is more stable to thermal denaturation than its counterpart from lobster muscle [Harris et al. (1980) Eur. J. Biochem. 108, 535-547]. Extra buried ionic bonds between subunits of the thermophilic enzyme make an important contribution to thermal stabilisation. Further stabilisation of the tetrameric enzyme is derived from additional hydrophobic interactions between the S-loops at the core of the tetramer. In the enzyme from the extreme thermophile Thermus aquaticus, which is even more thermostable, intersubunit ion pairs must also play a role but changes in interactions at the surface appear to be equally important. Thus additional hydrophobic interactions at the edge of subunit interfaces would prevent access of water to the interior of the molecule. Furthermore, the arrangement of charged residues on the surface of the T. aquaticus enzyme would allow maximal surface ion pair formation. The presence of surface ion pairs in other proteins correlates well with thermal stability [Perutz, M. F. and Raidt, H. (1975) Nature (Lond.) 255, 256-258] and would provide a general stabilising influence on the subunit in this case. [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]

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]

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]

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]

Initial velocity kinetics of the reverse direction showed that the saccharopine dehydrogenasecatalyzed reaction was consistent with a mechanism that the addition and release of reactants to and from the enzyme follow an obligatory order. The product inhibition studies in the forward direction also revealed kinetic patterns consistent with the ordered mechanism where the sequence of addition of substrates being NAD+ and saccharopine, and that of release of products being lysine, α-ketoglutarate and NADH. The various kinetic parameters determined at pH 6.8 and 22° are aisc described. [ABSTRACT FROM AUTHOR]

Penicilloylated proteins which are found as an impurity in 6-aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the point polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carded out in 6-aminopenicillanic acid preparations, since even a large excess of 6-aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme. In order to avoid the contamination of 6-aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process. [ABSTRACT FROM AUTHOR]

Absorption spectroscopy and circular dichroism measurements have been used to elucidate thc mechanism of lysozyme inactivation on photo-oxidation of the methionyl residues to methionine sulphoxide. The monosulphoxide derivative, which is formed by irradiation of the protein in aqueous solution, displayed spectral properties very similar to those of native lysozyme and retained about 54% enzyme activity. This reduced catalytic efficiency appeared to be due to limited conformational changes of the protein molecule. Striking differences were found, on the contrary, between the optical parameters of native lysozyme and of the di-sulphoxide derivative, which is prepared by irradiation in acetic acid solution and is almost devoid of enzyme activity. Studies on the unirradiated lysozyme demonstrated that this extensive denaturation was caused by the action of the protic solvent used. Whereas the acid-induced denaturation was reversible in the case of unmodified lysozyme, the photo-oxidation of the two methionyl residues irreversibly blocked the molecule in a largely disordered form. These results are discussed with respect to the location of the two methionines in the known atomic model of lysozyme. [ABSTRACT FROM AUTHOR]

Bovine β-lactoglobulin-A was hydrolysed with trypsin to yield a mixture of peptides which would not precipitate with rabbit antibody against the native protein, but would still inhibit the antigen-antibody reaction. The hydro- lysate was fractionated by ion exchange chromatography and found to contain seven antigenically active fractions. Two of these fractions were found to be homogeneous peptides. Each inhibited the reaction of antigen with antibody against the intact protein in haemagglutination, flocculation and passive cutaneous anaphylaxis reaction in guinea-pigs. One of the peptides had a molecular weight of 950 and the other had a molecular weight of 575. Both contained about equal numbers of polar and apolar amino acids. [ABSTRACT FROM AUTHOR]