Cross-reactivity of the membrane teichoic acid of Streptococcus faecalis N.C.I.B. 8191 in antipneumococcal type XII and type XVI sera

The membrane teichoic acid (MTA) precipitates heavily in the equine and rabbit antipneumococcal (anti-Pn) type XII sera tested. MTA contains multiple kojibiosyl residues and this reaction confirms the presence of kojibiosyl residues in the capsular polysaccharide, S XII, the antigenic determinant of pneumococcal type XII. MTA also precipitates one-third of the antibodies in an anti-Pn XVI serum: the poly(glycerol phosphate) residues of MTA may be involved in this reaction as well as d-glucose residues. Precipitation of MTA in anti-Pn XII is strongly inhibited by kojibiose, but the reaction in anti-Pn XVI is unaffected.     

Influence of the conserved active site residues of histidyl tRNA synthetase on the mechanism of aminoacylation reaction

The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase from Escherichia coli, Thermus thermophilus and Staphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mg²⁺ ions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction for E. coli, T. thermophilus and S. aureus are calculated using the combined ab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.     

Changes in Lysozyme due to Interaction with Vaporized Hexanal

In order to elucidate the mechanism of the alteration of proteins induced by vaporized aldehydes, unmodified and chemically-modified lysozymes were exposed in the solid state to vaporized hexanal at 50°C and 5.8 or 75% relative humidity (RH). On exposure at 75%RH, the unmodified lysozyme exhibited polymerization, browning, loss of solubility, fluorescence production and impairment of lysine, tryptophan and methionine residues. Methionine residues seemed to be oxidized to methionine sulfoxide residues. The polymerization did not proceed at 5.8RH. All the above alterations were almost completely prevented by the removal of oxygen from the reaction cells. Acetylation of lysozyme retarded these alterations fairly well except that the impairment of tryptophan residues was unaffected.

On the basis of all the results it is suggested that at the first step the concerned reaction essentially requires hexanal derivatives such as peroxyhexanoic acid and/or related radicals induced through the reaction with oxygen. The second step seems to consist at least of two routes which are independent of each other and require water. One route is assumed to be an amino-carbonyl reaction involving lysine residues. The other route seems responsible for the attack on tryptophan and methionine residues through oxidation involving the radicals.     

Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product

Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.     

Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction

The pH dependence of the reaction of various renins was investigated using sheep angiotensinogen as a substrate. Human renin showed two separate peaks, but rat and mouse Ren1 renins showed one peak with a shoulder. A comparison of the predicted subsite residues of human renin with those of rat and mouse Ren1 renins revealed that Arg82, Ser84, Thr85, Ala229, and Thr312 are unique in the human sequence. We examined the possible importance of these residues in the unique pH profile of the human renin reaction by replacing these residues with the corresponding residues of rat renin. The replacement of Ser84 of human renin with Gly changed the pH dependence of the reaction to one peak, similarly to rat and mouse Ren1 renins. Other mutant human renins kept two separate peaks, similarly to wild-type human renin. These results indicate that Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.     

N-terminal modification of proteins—a review

The first efforts to modify the terminal α-amino groups of proteins without reaction of the ?-amino groups of lysine residues made use of their lower pK values. A pH below 7 favors modification of weaker bases, since the stronger bases, although more reactive, are protected to an even greater extent by protonation. Unfortunately, this approach only favors modification of terminal over side-chain amino groups to a limited extent. N-Terminal serine and threonine residues may be selectively acylated on the amino group by an acyl transfer reaction after a peptide has been selectively acylated on its hydroxyl groups. This approach is severely limited by the need for the peptide to be stable to the acidic and anhydrous conditions necessary for selective O-acylation, and to the alkaline conditions necessary for removing the remaining O-acyl groups. Terminal serine and threonine residues may also be selectively oxidized by periodate, since this reaction is a thousand-fold faster than other oxidations of periodate, e.g., of 1,2-diols or disulfides. Further, it forms glyoxyloyl groups, which may be converted into terminal glycine residues by transamination. The last observation provided the basis for the one general modification of N-terminal residues, namely their conversion into 2-oxoacyl groups by reaction of the α-amino group with glyoxylate, a reaction catalysed by a bivalent cation, e.g., Cu²⁺, and a base, e.g., acetate. Participation of the neighboring peptide bond in the reaction ensures specificity of the reaction for the N-terminus. Scission of the N-terminal residue is possible after such a transamination; hence residues may be removed from the N-terminus under nondenaturing conditions. Other exploitations of transamination may be developed.     

Hydroxyl radical mediated damage to proteins, with special reference to the crystallins.

Oxidative modification of the eye lens proteins, the crystallins, is known to cause protein cross-links and aggregates which lead to lens opacification or cataracts. We focus attention here on oxidative damage occurring in crystallins and some "control" proteins upon reaction with the hydroxyl radical (.OH) which, in the lens, is generated by photosensitization or by the Fenton reaction. In the present study, we have synthesized and used the bishydroperoxide I as a "photo-Fenton" reagent, in order to photolytically generate pure .OH, free of other oxyradicals. Our findings are the following: (i) Trp residues are oxidized by .OH to N-formylkynurenine and related compounds, but this in itself does not lead to covalent aggregation of the protein. (ii) Tyr residues react with .OH, but apparently do not produce dihydroxyphenylalanine or bityrosine. Nor do protein cross-links occur as a result. (iii) Oxidation of His residues appears to be obligatory for protein cross-linking. Histidine-free proteins do not form high molecular weight products upon reaction with .OH. Protection of His residues by adduct formation in other proteins inhibits cross-linking. (iv) Lys residues seem to participate in the cross-linking reaction. Protection of the Lys residues by maleylation of the protein inhibits cross-linking. (v) The oxidized protein is more acidic in nature than the parent, and it might have altered conformational features.     

Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

The condensation step of fatty acid elongation is the addition of a C₂ unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies

DNA polymerases play a crucial role in the cell cycle due to their involvement in genome replication and repair. Understanding the reaction mechanism by which these polymerases carry out their function can provide insights into these processes. Recently, the crystal structures of human DNA polymerase lambda (Pollambda) have been reported both for pre- and post-catalytic complexes [García-Díaz et al., DNA Repair 3 (2007), 1333]. Here we employ the pre-catalytic complex as a starting structure for the determination of the catalytic mechanism of Pollambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg(2+) and Mn(2+) as the catalytic metals. In both cases the reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated by one of the conserved Asp residues (D490) in the active site before the incorporation of the nucleotide to the nascent DNA chain. A significant charge transfer is observed between both metals and some residues in the active site as the reaction proceeds. The optimized reactant and product structures agree with the reported crystal structures. In addition, the calculated reaction barriers for both metals are close to experimentally estimated barriers. Energy decomposition analysis to explain individual residue contributions suggests that several amino acids surrounding the active site are important for catalysis. Some of these residues, including R420, R488 and E529, have been implicated in catalysis by previous mutagenesis experiments on the homologous residues on Polbeta. Furthermore, Pollambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Polbeta, both of which are linked to cancer. In addition, residues R386, E391, K422 and K472 appear to have an important role in catalysis and could be a potential target for mutagenesis experiments. There is partial conservation of these residues across the Pol X family of DNA polymerases.     

Reactions of beta-propiolactone with nucleobase analogues, nucleosides, and peptides: implications for the inactivation of viruses

β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses.     

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.     

Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase

The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction. This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS. Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism. The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other. The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center. Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex.     

Attachment of peptide building blocks to proteins through tyrosine bioconjugation

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.     

Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, ¹H- and ¹³C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

Reaction of myosin with salicylaldehyde I. The effect of salicylaldehyde on the physicochemical properties of myosin

The specificity and nature of the reaction between salicylaldehyde and myosin and the effect of salicylalation on the molecular parameters of myosin were studied. The following observations were made.

1. 1. The reaction of salicylaldehyde with the lysyl residues of myosin is specific, since no salicylaldehyde is bound if the lysyl residues of myosin are trinitrophenylated.

2. 2. Salicylaldehyde is bound by myosin through the formation of an azomethine linkage (Schiff's base). This was established from the measured difference absorption spectrum of the myosin-salicylaldehyde complex.

3. 3. Three groups of lysyl residues can be distinguished with respect to the reaction with salicylaldehyde, namely, (a) residues with high association constant (K_ass = 1.8 ± 0.9·10⁵ M^-1), (b) residues with moderate association constant (K_ass = 2.2·10³ M^-1) and (c) residues that react with salicylaldehyde only after the denaturation of the protein. Their numbers could be estimated as 10 ± 5, 130 ± 5 and 260 ± 5 per mole myosin, respectively. The first group of residues was found to be absent from heavy and light meromyosin, the proteolytic fragments of myosin.

4. 4. The reaction is reversible. The complex formation rate constant, evaluated from the formula for second order reaction, is 2.2 sec^-1·M^-1, and the decomposition rate constant for first order reaction is 1.1·10^-3 sec^-1 at 22°.

5. 5. The reaction is pH dependent, the reaction yield increasing at higher pH.

6. 6. The solubility of myosin at low ionic strength decreases with increasing degree of salicylalation at slightly alkaline pH.

7. 7. The intrinsic viscosity of myosin does not change on salicylalation.

8. 8. A second peak due to polymerization appears on the sedimentation profile of the protein if more than 70 lysyl residues are salicylalated per mole of myosin.

Light-Induced Damage of Histidine Residues in Photosystem II Reaction Center D1/D2/cyt b559 Complex

Histidine residue content of photosystem Ⅱ reaction center D1/D2/cytochrome b559 complex decreased by about 26% after illumination. The result suggests that some histidine residues are damaged by illumination. The damage of histidine residues may be related to the changes of the spectra properties during the incubation in the dark following preillumination of the reaction center complex.     

Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

The self-association reaction of denatured staphylococcal nuclease fragments, urea-denatured G88W110, containing residues 1-110 and mutation G88W, and physiologically denatured 131-residue Delta 131 Delta, have been characterized by NMR at close to neutral pH. The two fragments differ in the extent and degree of association due to the different sequence and experimental conditions. Residues 13-39, which show significant exchange line broadening, constitute the main association interface in both fragments. A second weak association region was identified involving residues 79-105 only in the case of urea-denatured G88W110. For residues involved in the association reaction, significant suppression of the line broadening and small but systematic chemical shift variation of the amide protons were observed as the protein concentration decreased. The direction of chemical shift change suggests that the associated state adopts mainly beta-sheet-like conformation, and the beta-hairpin formed by strands beta 2 and beta 3 is native-like. The apparent molecular size obtained by diffusion coefficient measurements shows a weak degree of association for Delta 131 Delta below 0.4 mM protein concentration and for G88W110 in 4 M urea. In both cases the fragments are predominantly in the monomeric state. However, the weak association reaction can significantly influence the transverse relaxation of residues involved in the association reaction. The degree of association abruptly increases for Delta 131 Delta above 0.4 mM concentration, and it is estimated to form a 4 to 8 mer at 2 mM. It is proposed that the main region involved in association forms the core structure, with the remainder of residues largely disordered in the associated state. Despite the obvious influence of the association reaction on the slow motion of the backbone, the restricted mobility on the nanosecond timescale around the region of strand beta 5 is essentially unaffected by the association reaction and degree of denaturation.