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PubMed1999

Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein.

Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.

Adhesins, Bacterial↗

PubMed2005

Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli.

BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

Bacterial Proteins↗

PubMed1976

Implications of electrostatic potentials on ribosomal proteins.

Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developed by the 16S and 23S rRNA fractions. Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations. The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed accordingly.

Bacterial Proteins↗

PubMed1999

Complementary DNA cloning and characterization of ferredoxin localized in bundle-sheath cells of maize leaves.

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of -423 and -406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Amino Acid Sequence↗

PubMed1979

[Interaction of rat liver dexamethasone-receptor complexes with DNA].

Rat liver glucocorticoid-receptor complexes (GRC) acquire the ability to bind to DNA in a high affinity manner after activation by heating or precipitation with (NH)2SO4. DNA is practically non-saturable by GRC in low salt buffers as well as in 0.15 M NaCl-containing buffer, although in the latter case the binding decreases approximately 3--5 times. GRC bind to homo- and heterologous prokaryotic DNA in a similar way; in both cases an addition of KCl (up to 0.15 M) to the medium is followed by the same decrease of the binding. This data suggest that the association of GRC with DNA observed in vitro is not accompanied by "recognition" of any certain DNA site. Besides DNA, activated GRC can associate with other polymers, charged positively (DEAE-cellulose) or negatively (RNA, polyvinylsulfate). GRC interact very weakly with neutral compounds of the cellulose type but are strongly adsorbed on hydroxyapatite. Hence the activated GRC can be considered as an amphoteric protein. Salt solutions provoke dissociation of the GRC-DNA triple complexes: a complete dissociation is observed in the presence of 0,4 M NaCl or 0,4 M sodium phosphate buffer (pH 6,9). Sodium phosphate buffer also elutes GRC from other sorbents such as DEAE-cellulose or hydroxyapatite. No significant dissociation of the GRC-DNA complexes is observed at sucrose concentration up to 2 M. The data obtained are indicative of an essential role of electrostatic forces for the interaction of GRC with DNA. The non-ionic detergent Triton X-100 at a concentration as low as 0,05% completely destroys the GRC-DNA triple complexes. The models explicating the selectivity of the genome activation by GRC without their "recognition" of any specific DNA sequences are proposed.

Animals↗

PubMed1999

Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study.

The critical functions of the HIV-1 nucleocapsid protein NCp7 in genomic RNA packaging and reverse transcription, essentially rely on interactions with nucleic acids. A significant progress in the knowledge of these interactions has been recently achieved with the NMR-derived structures of NCp7 derivatives in complex with two short sequences of the HIV-1 psi packaging signal, namely ACGCC and the stem-loop 3 (SL3) motif. To further identify the key nucleotides in the formation of both NCp7-d(ACGCC) and NCp7-SL3 complexes, we quantitatively analyzed by steady-state and time-resolved fluorescence, the interaction of NCp7 with d(ACGCC) and SL3 mutants where each nucleotide in interaction with the protein has been systematically substituted. Moreover, by using several NCp7 derivatives, we investigated the contributions of Phe16, Trp37, and Trp61, and the various NCp7 domains, in the binding process. The binding of NCp7 appeared essentially driven by the interaction of the zinc finger domain and notably Trp37 with a G residue, irrespective of its location in the oligonucleotide. The involvement of Trp37 in the binding process depended on its location in the C-terminal finger motif and the proper folding of this motif. Phe16 in the N-terminal finger motif also strongly contributed to the binding energy, while in contrast, Trp61 in the C-terminal domain only marginally interacted with the oligonucleotides. The stem-loop structure of SL3 stabilized the binding of NCp7 by about -7 kJ/mol (at 0.1 M NaCl) by favoring the electrostatic binding of both N- and C-terminal domains. Finally, we found that NCp7 bound to nucleic acid single-stranded regions with the following preference: X(i)()TGX(j)() > X(i)()GXGX(j)() approximately X(i)()TXGX(j)() > X(i)()GX(j)() >> X(i)()X(j)(), where X corresponds to either A or C. This implies that recognition of nucleic acids by NCp7 may be achieved by a limited number of sites, and hence, no strong affinities are required in order to get a selective binding.

Amino Acid Sequence↗

PubMed1982

Stability decrease of RNA double helices by phenylalanine-, tyrosine- and tryptophane-amides. Analysis in terms of site binding and relation to melting proteins.

The amides of L-phenylalanine, L-tyrosine and L-tryptophane decrease the melting temperatures tm of poly(A)*poly(U) and poly(I)*poly(C) double helices at low concentrations (1 mM), whereas high concentrations finally lead to an increase of tm. This dependence of the tm-values upon the ligand concentration can be represented quantitatively by a simple site binding model, providing binding parameters for the interaction between the amides and the nucleic acids both in the double- and the single-stranded conformation. According to these data the affinity to the single strands is higher than that to the double strands and increases in the series Phe less than Tyr less than Trp. The binding constants decrease with increasing salt concentration as expected for an interaction driven by electrostatic attraction. However, part of the interaction is also due to stacking between the aromatic amides and the nucleic acid bases. The present results indicate a direct correlation between the presence of aromatic amino acids at the binding site of helix destabilising proteins and the properties of simple derivatives of these amino acids. Furthermore the results suggest that very simple peptides containing aromatic amino acids served as a starting point for the evolution of helix destabilising proteins.

Chemical Phenomena↗

PubMed2003

Binding surface mapping of intra- and interdomain interactions among hHR23B, ubiquitin, and polyubiquitin binding site 2 of S5a.

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.

Amino Acid Motifs↗

PubMed1980

Effect of hydrostatic pressure upon ethidium bromide association with transfer ribonucleic acid.

The binding of ethidium bromide to yeast phenylalanine-specific transfer ribonucleic acid (tRNAPhe) has been investigated in the pressure range from 1 atm to 9 kbar in the presence of 100 mM sodium chloride and 10 mM magnesium chloride, pH 7.7. One high-affinity binding site for ethidium is present, with a dissociation constant of 2.4 X 10(-6) M at 1 atm and 22 degrees C. Binding to this site is enhanced with increasing pressure, the dissociation constant reaching 2.9 X 10(-7) M at 2 kbar. Pressure also promotes the binding of ethidium to lower affinity sites of tRNAPhe. The standard volume change upon complex formation is found to be 25.6 +/- mL/mol for the first ethidium bound. If sodium is replaced by lithium in the buffer, the standard volume change is 23.3 +/- 0.5 mL/mol. We conclude that decrease of the electrostatic repulsion in the negatively charged tRNAPhe by binding of the positively charged ethidium is the main cause of the relatively large volume decrease upon complex formation. The electrostatic repulsion that must be present in this case, as well as in other nucleic acids, implies that intercalating binding sites are of the "soft" type as previously defined [Torgerson, P.M., Drickamer, H.G., & Weber, G. (1979) Biochemistry 18, 3079]. Model studies by others of the binding site characteristics are in agreement with this concept.

Chemical Phenomena↗

PubMed2005

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors.

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.

Acetylcholine↗

PubMed1997

Modulation of nucleic acid structure by ligand binding: induction of a DNA.RNA.DNA hybrid triplex by DAPI intercalation.

The aromatic diamidine, DAPI (4',6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid triplex that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT).poly(rA).poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA).poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT).poly(rA).poly(dT) triplex melts via two distinct transitions: initial conversion of the triplex to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT).poly(rA).poly(dT) triplex, an observation consistent with the preferential binding of the ligand to the triplex versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly(dT).poly(rA).poly(dT) triplex to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA).poly(dT) duplex. Our flow linear dichroism and viscometric data are consistent with an intercalative mode of binding when DAPI interacts with both the poly(dT).poly(rA).poly(dT) triplex and the poly(rA).poly(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT).poly(rA).poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.

Calorimetry, Differential Scanning↗

PubMed1984

Poly(8-bromodeoxyadenylic acid): properties of the polymer and contrast with the ribopolynucleotide analogue.

Introduction of the bulky 8-bromo substituent into adenine residues of polynucleotides has strikingly different consequences in the deoxy- and ribopolynucleotide series. Poly(r8BrA) was found in earlier studies to form a very stable double-helical self-structure but not to undergo interaction with potentially complementary polynucleotides. We find that poly(d8BrA), in contrast, does not form an ordered self-structure in 0.1 M Na+ but appears to exist as an electrostatically expanded rigid rod with unusual circular dichroism (CD) properties at very low ionic strength. The deoxy polymer, moreover, readily forms double helices with either deoxy or ribo pyrimidine polynucleotides, studied by UV, CD, and IR spectroscopy. These complexes are destabilized, relative to those formed by poly(dA), possibly because energy is needed to convert the purine residues from a more stable syn to an anti conformation, required for heteroduplex formation. The CD spectrum of (d8BrA)n X (dT)n is similar to that of B DNA. The deoxy-ribo hybrids (d8BrA)n X (rU)n and (d8BrA)n X (rBrU)n have CD spectra resembling those of A DNA or RNA. Unlike other deoxy-deoxy pairs (d8BrA)n X (dBrU)n, however, has a CD spectrum resembling RNA and other helices having the A form.

Chemical Phenomena↗

PubMed2004

Structure and mechanism of mRNA cap (guanine-N7) methyltransferase.

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.

Alanine↗

PubMed1994

On the conformational stability of oligonucleotide duplexes and tRNA molecules.

Thermodynamic experiments provide a wealth of data about the conformational stability, viz., the free energy difference (delta G) between folded and unfolded states of DNA/RNA duplexes. However, there is no acceptable view about how the various non-covalent forces contribute individually to the observed stability. In particular, the role of the hydrophobic force is not clearly known. In this paper we quantitatively enumerate the stability factors, hydrogen bonding, base stacking, van der Waals, electrostatic, and hydrophobic interactions from the knowledge of the crystal structures of 15 DNA/RNA duplexes and two tRNA molecules, and translate them into free energy contributions to the stability of nucleic acid systems. Taking the experimental delta G values and computed component free energy terms for a set of duplexes, we set up multiple regression equations to predict their stabilities. After back-check and validity tests, we apply this model to predict delta G values for a large number of duplexes and two tRNA molecules (tRNAphe and tRNAasp). There is excellent agreement between the theoretical predictions and experimental observations. The considered duplexes with four to 16 base-pairs and the tRNA molecules have delta G values in a narrow range, 5-20 kcal mol-1, a range seen in a variety of globular proteins. There is no relationship between delta G and N, the number of nucleotides in the molecule. Base-stacking, hydrogen bonding and van der Waals factors contribute significantly, whereas hydrophobic and electrostatic factors contribute, respectively, marginally and minimally. The major factor which gives sequence specificity is base-stacking. The new set of atomic solvation parameters (ASPs) derived to estimate hydrophobic free energy brings to light the dangers of using already available ASPs, which emphasize the role of the hydrophobic factor unrealistically.

Base Sequence↗

PubMed1995

Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions.

Base Composition↗

PubMed1995

The interaction of synthetic templates with eukaryotic DNA primase.

The template interaction of calf thymus and human placenta DNA primases has been investigated. Using oligothymidylates, we showed that a template consisting of ten monomeric units was the critical size for interaction with the enzyme. The hydrophobic effect is likely to be a major factor determining template recognition by the DNA primase. The correlation between the template affinity with the enzyme and the octanol-water hydrophobic scale confirms this suggestion. In addition, the electrostatic interaction between the phosphate group of the template and side chains on the enzyme probably increases template affinity. Using methylated poly(dA), we found that the first nucleotide base of the primer should be more hydrophobic than the corresponding nucleotide base of the template. A model for the mechanism of action of DNA primase is suggested on the basis of data presented in this study and previous findings. According to this model, (a) DNA primase binds ten nucleotides of the template; (b) the synthesis of primer up to the formation of a decamer occurs processively and competes with template binding of the enzyme; (c) the conditions under which the nucleotides are incorporated into the RNA product change during the extension of the primer, and these changes are responsible for switching from primase to polymerase activity. The template specificity of DNA primase is likely to play an important role in the initiation and regulation of DNA replication.

Base Sequence↗

PubMed2003

The long-range electrostatic interactions control tRNA-aminoacyl-tRNA synthetase complex formation.

In most cases aminoacyl-tRNA synthetases (aaRSs) are negatively charged, as are the tRNA substrates. It is apparent that there are driving forces that provide a long-range attraction between like charge aaRS and tRNA, and ensure formation of "close encounters." Based on numerical solutions to the nonlinear Poisson-Boltzmann equation, we evaluated the electrostatic potential generated by different aaRSs. The 3D-isopotential surfaces calculated for different aaRSs at 0.01 kT/e contour level reveal the presence of large positive patches-one patch for each tRNA molecule. This is true for classes I and II monomers, dimers, and heterotetramers. The potential maps keep their characteristic features over a wide range of contour levels. The results suggest that nonspecific electrostatic interactions are the driving forces of primary stickiness of aaRSs-tRNA complexes. The long-range attraction in aaRS-tRNA systems is explained by capture of negatively charged tRNA into "blue space area" of the positive potential generated by aaRSs. Localization of tRNA in this area is a prerequisite for overcoming the barrier of Brownian motion.

Amino Acyl-tRNA Synthetases↗

PubMed1975

The catalytic mechanism of amino acid:tRNA ligases. Synergism and formation of the ternary enzyme-amino acid-ATP complex.

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.

Adenosine↗