Thermodynamics of the interaction of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in aqueous solutions at 298.15K

The Gibbs energy interaction parameters of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in water, g(ES), were obtained from electromotive force (emf) measurements of the electrochemical cell without liquid junction and containing two ion-selective electrodes (ISE): K-ISEmid R:RbCl(m(E))mid R:ISE-Cl and K-ISEmid R:RbCl(m(E)),saccharide (m(S))mid R:ISE-Cl, at 298.15K. The enthalpy interaction parameters of RbCl with these monosaccharides in water, h(ES), are determined according to the McMillan-Mayer theory from the measurements of the enthalpies of mixing of aqueous RbCl solutions with aqueous monosaccharide solutions, as well as the enthalpies of dilution of RbCl and monosaccharide solutions in pure water at 298.15K by a calorimetric method. Furthermore, the entropy interaction parameters, s(ES), can be evaluated through g(ES) and h(ES). The results suggest that the electrostatic interactions of these monosaccharides with RbCl in water are predominant compared with structural interactions, and these parameters are controlled primarily by the stereochemical structure of the monosaccharides in water.     

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids

The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.     

Activity coefficients of the electrolyte and the amino acid in water + NaNO(3) + glycine and water + NaCl + DL-methionine systems at 298.15 K

The activity coefficients at 298.15 K of glycine in water + NaNO(3) + glycine system and dl-methionine in water + NaCl + dl-methionine system are reported. The measurements were performed in an electrochemical cell with two ion selective electrodes, a cation and an anion ion selective electrode, each versus a double junction reference electrode. The concentrations of the electrolytes and the amino acids studied covered up to 1.0 molality electrolyte, 2.4 molality glycine and 0.2 molality dl-methionine. The results of the activity coefficients of glycine are compared with the activity coefficients of glycine in water + NaCl + glycine and water + KCl + glycine systems, obtained from the previous studies. The results show that the nature of both the cation and the anion of an electrolyte have significant effects on the activity coefficient of glycine in aqueous electrolyte solutions. The results also show that there are attractive interactions between the molecules of glycine and NaNO(3) and repulsive interactions between the molecules of dl-methionine and NaCl.     

Binding of berberine to human telomeric quadruplex - spectroscopic, calorimetric and molecular modeling studies

This study examines the characteristics of binding of berberine to the human telomeric d[AG(3)(T(2)AG(3))(3)] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry, we found that the binding affinity of berberine to the human telomeric quadruplex is 10(6). The complete thermodynamic profile for berberine binding to the quadruplex, at 25 degrees C, shows a small negative enthalpy (DeltaH) of -1.7 kcal.mol(-1), an entropy change with TDeltaS of +6.5 kcal.mol(-1), and an overall favorable free energy (DeltaG) of -8.2 kcal.mol(-1) .Through the temperature dependence of DeltaH, we obtained a heat capacity (DeltaC(p)) of -94 (+/- 5) cal.mol(-1).K(-1). The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex-berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the pi-pi interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DeltaC(p)) values for quadruplex-berberine models are -89 and -156 cal.mol(-1).K(-1).     

Ouabain-insensitive Na+ stimulation of an Mg-2+ -dependent ATPase in kidney tissue.

1. Freshly prepared microsomal fractions of the outermost cortex of guinea pig kidney show an Mg-2+-dependent ATPase activity which is partially inhibited by 100 mM NaCl, LiCl, KCl, RbCl, CsCl, NH4Cl or choline chloride. 2. If the microsomal preparation is aged by storage at 4 degrees C for 10-15 days, the Mg-2+-dependent activity shows stimulation by Na-+ and Li-+ but not by K-+, Rb-+, Cs-+, NH4-+ or choline. 3. Stimulation is similar with sodium salts of Cl-minus, HCO3-minus, CH3COO-minus, BR-minus, SO4-2-minus or methylsulphonate. 4. Stimulation is insensitive to 1 mM and 10 mM ouabain. 5. Stimulation is unaltered by the presence of 0.5 mM ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetracetic acid. 6. Stimulation is 100% inhibited by 2 mM ethacrynic acid, a concentration which inhibits only 30% of the Mg-2+-dependent ATPase and 50% of the (Na-++K-+)-stimulated ATPase. 7. Some of these characteristics coincide with those of an ouabain-resistant, K-+-independent, ethacrynic acid-sensitive mode of Na-+ extrusion out of guinea pig kidney cortex cells.     

Mechanism,mutagenesis, and chemical rescue of a beta-mannosidase from cellulomonas fimi

The chemical mechanism of a retaining beta-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced. Studies with a series of aryl beta-mannosides of vastly different reactivities (pK(a)(lg) = 4-10) allowed kinetic isolation of the glycosylation and deglycosylation steps. Substrate inhibition was observed for all but the least reactive of these substrates. Br?nsted analysis of k(cat) revealed a downward breaking plot (beta(lg) = -0.54 +/- 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol. The pH dependence of k(cat)/K(m) follows an apparent single ionization of a group of pK(a) = 7.65 that must be protonated for catalysis. The tentative assignment of E429 as the acid-base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of beta-mannosyl azide and beta-mannosyl fluoride, respectively. A pH-dependent chemical rescue of E429A activity is also observed with citrate. Substantial oxocarbenium ion character at the transition state was demonstrated by the alpha-deuterium kinetic isotope effect for Man2A E429A of alpha-D(V) = 1.12 +/- 0.01. Surprisingly, this isotope effect was substantially greater in the presence of azide (alpha-D(V) = 1.166 +/- 0.009). Likely involvement of acid/base catalysis was revealed by the pH dependence of k(cat) for Man2A E429A, which follows a bell-shaped profile described by pK(a) values of 6.1 and 8.4, substantially different from that of the wild-type enzyme. The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding "glycosynthase" activities. The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (DeltaDeltaG() = 5.1 kcal mol(-1)) was probed using a 2-deoxymannose substrate. This value, surprisingly, is comparable to that found from equivalent studies with beta-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position. Modes of stabilizing the mannosidase transition state are discussed.     

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.     

Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.     

The effects of lithium, rubidium, cesium and magnesium ions on the close packing of persistence-length DNA fragments

By application of scaled particle theory to persistence-length DNA fragments in sedimentation-equilibrium at speeds high enough to maintain close packing, the range of interhelical electrostatic repulsion was evaluated with LiCl, RbCl, CsCl, and MgCl2 as supporting electrolytes. Analysis of the data in terms of the Zimm cluster function confirmed that the net interaction between helices is purely repulsive in all cases. At constant ionic strength the electrostatic radius of the rod-like DNA decreases as the counterion changes from Li+ to Rb+ to Cs+. In contrast to univalent counterions, electrostatic radius increases with Mg2+ concentration, except at very low (mM) MgCl2 concentrations. All solutions undergo a reversible transition to a turbid, optically anisotropic phase at a slightly salt-dependent, critical DNA concentration, as observed previously for NaDNA.     

A suboptimal algorithm for de novo peptide sequencing via tandem mass spectrometry.

Tandem mass spectrometry has emerged to be one of the most powerful high-throughput techniques for protein identification. Tandem mass spectrometry selects and fragments peptides of interest into N-terminal ions and C-terminal ions, and it measures the mass/charge ratios of these ions. The de novo peptide sequencing problem is to derive the peptide sequences from given tandem mass spectral data of k ion peaks without searching against protein databases. By transforming the spectral data into a matrix spectrum graph G = (V, E), where |V| = O(k(2)) and |E| = O(k(3)), we give the first polynomial time suboptimal algorithm that finds all the suboptimal solutions (peptides) in O(p|E|) time, where p is the number of solutions. The algorithm has been implemented and tested on experimental data. The program is available at http://hto-c.usc.edu:8000/msms/menu/denovo.htm.     

The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.     

A comparison of thermal characteristics and kinetic parameters of trehalases from a thermophilic and a mesophilic fungus

Trehalases from a thermophilic fungus Thermomyces lanuginosus (M(r) 145 kDa) and a mesophilic fungus Neurospora crassa (M(r) 437 kDa) were purified to compare their thermal characteristics and kinetic constants. Both trehalases were maximally active at 50 degrees C, had an acidic pH optimum and were glycoproteins (20% and 43%, w/w, carbohydrate content for T. lanuginosus and N. crassa, respectively). At their temperature optimum, their K(m) was similar (0.57 and 0.52 mM trehalose, for T. lanuginosus and N. crassa, respectively) but the V(max) of N. crassa enzyme was nine times higher than of T. lanuginosus enzyme. The catalytic efficiency, k(cat)/K(m), for N. crassa trehalase was one order of magnitude higher (6.2 x 10(6) M(-1) s(-1)) than of T. lanuginosus trehalase (4 x 10(5) M(-1) s(-1)). At their T(opt) (50 degrees C), trehalase from both sources exhibited similar thermostability (t(1/2)6 h). The energy of activation, E(a), for T. lanuginosus trehalase was 15.12 kcal mol(-1) and for N. crassa trehalase it was 9.62 kcal mol(-1). The activation energy for thermal inactivation for the N. crassa enzyme (92 kcal mol(-1)) was two-fold higher than for the T. lanuginosus enzyme (46 kcal mol(-1)). The present study shows that the trehalase of N. crassa is not only more stable but also a better catalyst than the T. lanuginosus enzyme.     

重组菌产碱性果胶酯裂解酶酶学性质研究

利用盐析-透析-色谱流程建立快速高效纯化工程菌E.coli JM109(pHsh PL)所产碱性果胶酯裂解酶(PL)的方法,纯化后酶达到电泳纯,比酶活为1079U/mg.重组菌所产PL酶促反应适宜的pH为9～10,适宜温度为50～66 ℃,与酶基因来源野生菌所产PL相比,重组菌所产PL适宜pH范围有所扩大,并保持了野生菌PL的热稳定性.通过金属离子种类、浓度及存在时间对PL酶活力影响考察发现:在考察的离子中除Mg2 对酶活有较好的促进作用外,其余对重组菌PL均有抑制作用,其中Fe2 对酶活力抑制作用最强.该酶的Km值为20.93 mg/L,Vmax为105.3 μmol/min,反应活化能Ea为21.74 kJ/mol.对重组菌所产PL热稳定动力学进行分析,发现有底物情况下的失活常数kd(0.02 min-1)小于无底物情况下的失活常数kd(0.0342 min-1),说明当酶与底物结合形成复合物时对酶活具有保护作用.利用HPLC-ESI-MS对重组菌所产PL酶解产物进行测定发现,产物含有不饱和二聚半乳糖醛酸(m/z 350.82)和不饱和三聚半乳糖醛酸(m/z 527.04),同时测定结果中没有发现不饱和半乳糖醛酸单体(m/z 175),可以初步推测重组菌PL不能以不饱和二聚半乳糖醛酸和不饱和三聚半乳糖醛酸为底物进一步裂解.     

A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.     

Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.     

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.     

Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis.

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity. This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site. To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A. The aglycone mutations caused an increase in both k(cat) and K(m) against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(cat) values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct. The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k(2) step, out of the active site.