Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

Energetics of a possible proton exit pathway for water oxidation in photosystem II

The crystal structure of photosystem II (PSII) at 3.0-A resolution suggests that titratable residues on the lumenal side of D1/D2 and PsbO form a polar channel, which might serve as a proton exit pathway associated with water oxidation on the Mn-cluster. With full account of protein environment, we calculated the pK(a) of these residues by solving the linearized Poisson-Boltzmann equation. Along the prospective proton channel, the calculated pK(a) of titratable residues (namely via D1-Asp61, D1-Glu65, D2-Glu312, D2-Lys317 D1-Asp59, D1-Arg64, PsbO-Arg152, and PsbO-Asp224) monotonically increase from the Mn-cluster to the lumenal bulk side. We suggest that these residues form the exit pathway guiding protons, which are released at the Mn-cluster as a product of water oxidation, in an exergonic process out of PSII. Upon the S2 to S3 transition, CP43-Arg357 showed a dramatic deprotonation of ca. one H(+), suggesting that this residue is coupled to the redox states of the Mn-cluster and the tyrosine Y(Z). The calculated pK(a) values of 4.2-4.4 for D2-Glu312 and those of approximately 8-10.9 for D1-Asp59 and D1-Arg64 are indicative of the experimentally determined pK(a) values for inhibition of S-state transitions. Upon removal of the atomic coordinates of PsbO, the pK(a) of these residues are dramatically affected, indicating a significant role of PsbO in tuning the pK(a) of those residues in the proton exit pathway.     

Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase

Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Br?nsted base 3-quinuclidinone in water, and it is not clear that such Br?nsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Br?nsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic price for desolvation of thiol anions than for desolvation of the more strongly solvated oxygen anions.     

Mechanism of zinc coordination by point-mutated structures of the distal CCHC binding motif of the HIV-1 NCp7 protein

The kinetics of Zn(2+) binding by two point-mutated forms of the HIV-1 NCp7 C-terminal zinc finger, each containing tridentate binding motif HCC [Ser49(35-50)NCp7] or CCC [Ala44(35-50)NCp7], has been studied by stopped-flow spectrofluorimetry. Both the formation and dissociation rate constants of the complexes between Zn(2+) and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn(2+) binding paths due to three deprotonated states of the coordinating motif, acting as monodentate, bidentate, and tridentate ligands. For Ser49(35-50)NCp7 around neutral pH, binding preferentially occurs via the deprotonated Cys36 in the bidentate state also involving His44. The binding rate constants for the monodentate and bidentate states are 1 x 10(6) and 3.9 x 10(7) M(-)(1) s(-)(1), respectively. For Ala44(35-50)NCp7, intermolecular Zn(2+) binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 x 10(7) M(-)(1) s(-)(1). In both mutants, the final state of the Zn(2+) complex is reached by subsequent stepwise ligand deprotonation and intramolecular substitution of coordinated water molecules. The rate constants for the intermolecular binding paths of the bidentate and tridentate states of Ala44(35-50)NCp7 and of the tridentate state of Ser49(35-50)NCp7 are much smaller than expected according to electrostatic considerations. This is attributed to conformational constraints required to achieve proper metal coordination during folding. The dissociation of Zn(2+) from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn(2+)-bound bidentate and monodentate states, with rate constants of approximately 0.3 and approximately 10(3) s(-)(1), respectively, for Ser49(35-50)NCp7 and approximately 4 x 10(-)(3) and approximately 500 s(-)(1), respectively, for Ala44(35-50)NCp7.     

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Galactose mutarotase: pH dependence of enzymatic mutarotation

Here we report pH dependence of kinetic parameters for the mutarotation of alpha-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k(cat) and k(cat)/K(m) for the mutarotation of alpha-D-galactose were found to be 1.84 x 10(4) s(-1) and 4.6 x 10(6) M(-1) s(-1), respectively, at pH 7.0 and 27 degrees C. The corresponding values for alpha-D-glucose were 1.9 x 10(4) s(-1) and 5.0 x 10(5) M(-1) s(-1). Inasmuch as the value of k(cat)/K(m) for the reaction of alpha-D-galactose is 10 times that for alpha-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of alpha-D-glucose should not be diffusion controlled. Therefore, pH-rate profiles should not be distorted by diffusion. The k(cat) for the mutarotation of alpha-D-glucose is independent of pH. Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k(cat)/K(m) versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and -2 on the alkaline side. The values of pK(a) are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of alpha-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k(cat)/K(m) versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK(a) values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of alpha-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.     

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Effect of histidine 6 protonation on the active site structure and electron-transfer capabilities of pseudoazurin from Achromobacter cycloclastes

The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin [PACu(II)] contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II). The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction. The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified. Corresponding changes are also seen in the UV/vis spectrum. The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range. The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)). The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.     

pH dependence of the kinetic parameters for 3-oxo-delta 5-steroid isomerase. Substrate catalysis and inhibition by (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17-one

The pH-rate profiles for kcatobsd and (kcat/KM)obsd at 25.0 degrees C have been measured for 3-oxo-delta 5-steroid isomerase by using 5-androstene-3,17-dione (2), 5-pregnene-3,20-dione (3), and 5(10)-estrene-3,17-dione (4) as substrates. Results from the nonsticky substrate 4 suggest values for the pK of a catalytically important group on the free enzyme (pKE) of 4.57 and the pK of the same group in the enzyme-substrate complex of 4.74. For the sticky substrates 2 and 3, pKES is ca. 4.75 and 5.5, respectively. Analysis of the (kcat/KM)obsd vs. pH profile for 2 reveals that the intermediate E X S complex decomposes to products at a rate similar to its reversion to E + S. The pH-rate profile for inhibition of the isomerase by (3S)-spiro-[5 alpha-androstane-3,2'-oxiran]-17-one (7 beta) shows values for pKE of 4.75 and pKEI of 4.90. The similarity of the pH-rate profiles for isomerization of 4 and inhibition by 7 beta suggests that both reactions may be governed by the ionization state of the same carboxyl group of the enzyme.     

The ionization behavior of retinoic acid in aqueous environments and bound to serum albumin.

The ionization behavior of retinoic acid (RA) in an aqueous phase and when bound to bovine serum albumin was studied. Titrations of RA in the various phases were followed by monitoring the red shift in the absorption maximum of RA that occurred upon deprotonation. The apparent pK of RA was dependent on the concentration of this compound. At the concentration range 6-20 microM, the pK of RA in water had a value of approximately 8.0. As the concentration was decreased in the range 1-6 microM, the value of the pK decreased continuously. The lowest pK observed was approximately 6.0. It was concluded that RA in an aqueous phase at concentrations in the microM range, forms micelles, and that the values of the pK of RA monomers and micelles in water are less than 6.0 and 8.0, respectively. The presence of 0.15 M NaCl caused a decrease in the pK of RA micelles and lowered the value of the CMC. Titration of RA in the presence of bovine serum albumin revealed the presence of a heterogeneous population comprised of three distinct microenvironments for RA associated with this protein. Two populations of RA were found to undergo complete titration in the pH range 4-8. A third population became apparent at pH greater than 9.5.     

Transport of water and glycerol in aquaporin 3 is gated by H(+).

Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L(p), was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. The L(p) values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeability P(Gl), obtained from [(14)C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (sigma). For AQP3, water and glycerol interacted by competing for titratable site(s): sigma(Gl) was 0.15 at neutral pH but doubled at pH 6.4. The sigma values were smaller for polyols in which the -OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol(-1). We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites.     

Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein

In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 x 10(3) h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9-10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pK(a) of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pK(a) of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 alpha-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pK values and stabilizing the alpha-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.     

Proton equilibria in the manganese cluster of photosystem II control the intensities of the S(0) and S(2) state g approximately 2 electron paramagnetic resonance signals

We have studied the pH effect on the S(0) and S(2) multiline electron paramagnetic resonance (EPR) signals from the water-oxidizing complex of photosystem II. Around pH 6, the maximum signal intensities were detected. On both the acidic and alkaline sides of pH 6, the intensities of the EPR signals decreased. Two pKs were determined for the S(0) multiline signal; pK(1) = 4.2 +/- 0.2 and pK(2) = 8.0 +/- 0.1, and for the S(2) multiline signal the pKs were pK(1) = 4.5 +/- 0.1 and pK(2) = 7.6 +/- 0.1. The intensity of the S(0)-state EPR signal was partly restored when the pH was changed from acidic or alkaline pH back to pH approximately 6. In the S(2) state we observed partial recovery of the multiline signal when going from alkaline pH back to pH approximately 6, whereas no significant recovery of the S(2) multiline signal was observed when the pH was changed from acidic pH back to pH approximately 6. Several possible explanations for the intensity changes as a function of pH are discussed. Some are ruled out, such as disintegration of the Mn cluster or decay of the S states and formal Cl(-) and Ca(2+) depletion. The altered EPR signal intensities probably reflect the protonation/deprotonation of ligands to the Mn cluster or the oxo bridges between the Mn ions. Also, the possibility of decreased multiline signal intensities at alkaline pH as an effect of changed redox potential of Y(Z) is put forward.     

Long-range nature of the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B. agaradhaerens xylanase

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.     

Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26.

Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1). For both sites, a proton was released upon Cd(2+) binding. One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site. The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2). The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985-14991]. A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.     

pH dependent kinetic studies of lipoamide dehydrogenase catalysis.

1. Kinetic studies of lipoamide dehydrogenase and its modified enzymes catalyzing lipoamide oxidoreduction and ancillary reactions at various pH are compared. 2. The asymptotic kinetics of lipoamide oxidoreductions switch between the ping pong and ordered mechanisms by varying pH of the reactions. 3. pH-rate profiles of these reactions are bell-shaped suggesting the participation of 2 ionizable residues with pK values of 6.6 +/- 0.5 and above 8 respectively. 4. The unusually high pK value for the catalytic site histidine is attributed to its involvement in an ion-pair formation. 5. In the absence of the catalytic site histidine, the pH-rate profile for the lipoamide reduction of the photooxidized enzyme is no longer bell-shaped but it is similar to those of the transhydrogenation and NADH-oxidation of the native enzyme. 6. This implies the participation of a low-pK protonated group in these reactions.     

Substrate specificity and kinetic characteristics of angiotensin converting enzyme

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.