Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Ω loop. To determine whether the Ω loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold, indicating severe alteration in the binding area in W430A. The k_cat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Ω loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.     

Does the proposed DSE motif form the active center in the Hermes transposase?

Donor cleavage and strand transfer are two functions performed by transposases during transposition of class II transposable elements. Within transposable elements, the only active center described, to date, facilitating both functions, is the so-called DDE motif. A second motif, R-K-H/K-R-H/W-Y, is found in the site-specific recombinases of the tyrosine recombinase family. While present in many bacterial insertion sequences as well as in the eukaryotic family of mariner/Tc1 elements, the DDE motif was considered absent in other classes of eukaryotic class II elements such as P, and hAT and piggyBac. Based on sequence alignments of a hobo-like element from the nematode Caenorhabditis elegans, to a variety of other hAT transposases and several members of the mariner/Tc1 group, Bigot et al. [Gene 174 (1996) 265] proposed the presence of a DSE motif in hAT transposases. In the present study we tested if each of these three residues is required for transposition of the Hermes element, a member of the hAT family commonly used for insect transformation. While D402N and E572Q mutations lead to knock-out of Hermes function, mutations S535A and S535D did not affect transposition frequency or the choice of integration sites. These data give the first experimental support that D402 and E572 are indeed required for transposition of Hermes. Furthermore, this study indicates that the active center of the Hermes transposase differs from the proposed DSE motif. It remains to be shown if other residues also form the active site of this transposase.     

Does alpha-tocopherol interact with the active center of cytochrome P-450 in liver microsomes?

The interaction of alpha-tocopherol (alpha-T) and its synthetic derivative 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (PMC) with cytochrome P-450 system was studied in the rat liver microsomes. Spectral differentiations of type I, increase of NADPH oxidation rate and inhibition of 7-ethoxycoumarin deethylase in microsomes were observed only in the presence of PMC. The results obtained suggest that unlike alpha-T, PMC is effectively bound and metabolized by cytochrome P-450.     

Does the non-saturable cell entry apply to the charge-free form of amino acids?

The slow cellular entry shown by neutral alpha-amino acids at very high concentrations appears not to arise from diffusion of the totally uncharged species through the plasma membrane of the Ehrlich cell, judging from a similarity of the rates observed for the two conformational isomers of 1-amino-2-hydroxy-cyclohexane-carboxylic acid. One of these isomers provides in neutral solution 4 times as large a proportion of the charge-free species as the other, and 5 times the proportion calculated for alanine.     

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold.     

How can the aromatic side-chains modulate the conductance of the gramicidin channel? A new approach using non-coded amino acids.

In order to elucidate the role of the aromatic side-chains in the mechanism of transduction of monovalent cations through the channel of linear gramicidin, two series of analogues containing non-coded aromatic amino acids were synthesized. In the first series, the four tryptophans were replaced by either four L-3-(8-quinolyl)alanyl or four L-3-(4-quinolyl)alanyl residues and single channel conductance measurements showed that these substitutions led to a strong lowering of the channel conductance, which is attributed to a modification of the orientation of the aromatic side-chains due to an increase of their hydrophobicity. In the second series, the analogues contained both tryptophyl and naphthylalanyl residues in various amounts and positions. The single channel conductance data indicated that the conductance was mainly governed by the number of polar residues (Trp) and not by their positions. The conformational consequences of these results are discussed together with their influence on the energy profile of the gramicidin channel.     

Does the trimeric form of the Photosystem 1 reaction center of cyanobacteria in vivo exist?

When isolating Photosystem 1 from the thylakoid membranes of cyanobacteria, a multiplicity of photosystem complexes can be encountered, which has not yet been observed in any other phototrophic organisms: After solubilisation of thylakoid membranes with detergents, trimeric, dimeric and monomeric forms of Photosystem 1 can be separated. The question must now be answered, which of the stable Photosystem 1 forms is the functional form in vivo-monomeric or trimeric? The two possibilities are discussed, though we mainly present arguments for the existence of the trimeric form of Photosystem 1 in cyanobacterial thylakoid membranes.     

Does the anthropometric model influence whole-body center of mass calculations in gait?

Examining whole-body center of mass (COM) motion is one of method being used to quantify dynamic balance and energy during gait. One common method for estimating the COM position is to apply an anthropometric model to a marker set and calculate the weighted sum from known segmental COM positions. Several anthropometric models are available to perform such a calculation. However, to date there has been no study of how the anthropometric model affects whole-body COM calculations during gait. This information is pertinent to researchers because the choice of anthropometric model may influence gait research findings and currently the trend is to consistently use a single model. In this study we analyzed a single stride of gait data from 103 young adult participants. We compared the whole-body COM motion calculated from 4 different anthropometric models (Plagenhoef et al., 1983; Winter, 1990; de Leva, 1996; Pavol et al., 2002). We found that anterior-posterior motion calculations are relatively unaffected by the anthropometric model. However, medial-lateral and vertical motions are significantly affected by the use of different anthropometric models. Our findings suggest that the researcher carefully choose an anthropometric model to fit their study populations when interested in medial-lateral or vertical motions of the COM. Our data can provide researchers a priori information on the model determination depending on the particular variable and how conservative they may want to be with COM comparisons between groups.     

Occurrence of mycosporine-like amino acids in the red-tide dinoflagellate Alexandrium excavatum: UV-photoprotective compounds?

Water extracts of the red-tide dinoflagellate Alexandrium excavatumgrown at ‘high’ light intensity (200 µE m^–2s^–1) show a broad absorbance maximum in the UV regionof the spectrum (310–360 nm). Using TLC and reverse-phaseHPLC a series of mycosporine-like amino acids have been characterized:mycosporine-glycine (_max = 310 nm), palythine (_max = 320 nm),asterina-330 (_max = 330 nm), shinorine (_max = 334 nm), porphyra-334(_max= 334 nm), palythenic acid (_max = 337 nm) and the isomericmixture of usujirene and palythene (_max = 359 nm). From theobserved spectral changes during transference from ‘low’(20 µE m^–2 s^–1) to ‘high’ (200µE m^–2 s^–1) light intensities and vice versa,the series of compounds are supposed to be biogenically relatedto one another. The presence of these compounds in A.excavatumis discussed in relation to their possible role in the photoprotectionto deleterious UV radiation.     

Does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?

Thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated Sephadex G-200 particles. Lys in place of Gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it.     

Does water play a structural role in the folding of small nucleic acids?

Nucleic acid structure and dynamics are known to be closely coupled to local environmental conditions and, in particular, to the ionic character of the solvent. Here we consider what role the discrete properties of water and ions play in the collapse and folding of small nucleic acids. We study the folding of an experimentally well-characterized RNA hairpin-loop motif (sequence 5'-GGGC[GCAA]GCCU-3') via ensemble molecular dynamics simulation and, with nearly 500 micros of aggregate simulation time using an explicit representation of the ionic solvent, report successful ensemble folding simulations with a predicted folding time of 8.8(+/-2.0) micros, in agreement with experimental measurements of approximately 10 micros. Comparing our results to previous folding simulations using the GB/SA continuum solvent model shows that accounting for water-mediated interactions is necessary to accurately characterize the free energy surface and stochastic nature of folding. The formation of the secondary structure appears to be more rapid than the fastest ionic degrees of freedom, and counterions do not participate discretely in observed folding events. We find that hydrophobic collapse follows a predominantly expulsive mechanism in which a diffusion-search of early structural compaction is followed by the final formation of native structure that occurs in tandem with solvent evacuation.     

Does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?

Thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated Sephadex G-200 particles. Lys in place of Gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it.     

Are learning and attention related to the sequence of amino acids in ACTH/MSH peptides?

Learning and attention were examined in rats after injections of one of several molecules related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). The initial phase of the learning process was linearly related to the length of the peptide with the smallest fragment (MSH/ACTH 4–10) improving learning the most and the largest molecule (ACTH 1–24) exerting no effect. Later stages of the learning problem, which were sensitive to the attentional state of the organism, resulted in U-shaped relations with the length of the same peptides. Enhancement of attention was significant only for the MSH compounds. These data indicate that some behaviors may be influenced as a function of the redundant information contained in the molecule while other behaviors may be discretely related to the specific conformation of the molecule.     

Does an increase in reward affect the precision of the encoding of directional information in the honeybee waggle dance?

Apis mellifera foragers perform waggle dances to communicate the presence of highly desirable nectar sources to their forager-mates. Each waggle dance consists of several waggle-runs (straight movements of the dancer closely aligned on the comb surface) that carry spatial information that the dance followers can use to locate the food source being advertised. To address how this complex motor display responds to unpredictable fluctuations in its main triggering stimulus, i.e., sucrose stimulation, we analyzed the effects of an increase in reward on the direction of consecutive waggle-runs as well as other components of the waggle dance. Results show that a sudden increase in reward may increase the directional scatter among consecutive waggle-runs, especially those performed at the beginning of the dance. However, a simultaneous and rapid increase in the duration of the signal—together with a more regular alignment of the later waggle-runs within the signal— seems to compensate the initial increase in directional scatter so that the transfer of directional information remains effective. These results point out that the regulation of dance maneuvers depends on the dancers motivation to forage.     

Molecular orbital calculation for the model compounds of kainoid amino acids, agonists of excitatory amino acid receptors. Does the kainoid C4-substituent directly interact with the receptors?

Kainoid amino acids are agonists of the AMPA/kainate receptors and exhibit highly potent neuroexcitatory activity. From the results of extensive structure--activity relationship studies, we previously postulated that the C4-substituent of the kainoid amino acids interacts with an allosteric site of the glutamate receptor with electron-donating character. In order to investigate the mode of action in more detail, molecular orbital calculation for model compounds of the kainoid were performed. The results indicated that the HOMO energy level of the C4-substituent is involved in the potent neuroexcitatory activity, thus supporting our hypothesis.     

Contribution of an aspartate residue,D114, in the active site of clostridial glutamate dehydrogenase to the enzyme’s unusual pH dependence

Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH. This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein. The D114N mutant enzyme shows a higher optimum pH for activity than the wild-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant.