Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by the phospholipase A2 from Agkistrodon halys blomhoffii venom

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25 degrees C and the ionic strength of 0.1 in the presence of 3-33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the alpha-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine were studied by the tryptophyl fluorescence method at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of substrate (monomer) molecules, N = 10, 5 or 13 for N. naja atra apoenzyme and its Ca2+ complex, and N. naja kaouthia apoenzyme, respectively. The binding constant of the enzymes to the micelle, Kmic = 0.18-3.1 X 10(6) M-1, was 9-160 times greater than that to the monomeric substrate, Kmon = 2 X 10(4) M-1 (Teshima et al. (1981) J. Biochem. 89, 1163-1174). This was interpreted in terms of the presence of an additional substrate-binding site in the enzyme molecule. The binding constant of the enzyme-Ca2+ complex to the micelle was smaller than that for the apoenzyme over a wide range of pH. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.4 to 5.53 and 7.55 to 7.95. The pH dependence curve for the Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of only a single ionizable group from 7.25 to 7.55. The former ionizable group was assigned as Asp 49, to which Ca2+ can coordinate, and the latter as His 48 in the active site on the basis of the reported pK values of these ionizable groups in the apoenzyme and Ca2+ complex (Teshima et al. (1981) J. Biochem. 89, 13-20 and Teshima et al. (1982) J. Biochem. 91, 1777-1788). No participation of the alpha-amino group with a pK value of 8.55 was observed.     

Binding of N-acetyl-chitotriose to Asp 52-esterified hen lysozyme

The pH dependence of the binding constant of (GlcNAc)3 to Asp 52-esterified lysozyme was determined by the fluorescence technique. The pK values of Asp 101 in the modified lysozyme and its complex with (GlcNAc)3 were determined to be 4.5 and 3.6, respectively, at 25 degrees C and 0.1 ionic strength. This result is different from that obtained by Parsons and Raftery ((1972) Biochemistry 11, 1633--1638), who observed no pK shift of Asp 101. The macroscopic pK value of Asp 52 in intact lysozyme determined by them using the pH difference titration data of Asp 52-esterified lysozyme relative to intact lysozyme ((1972) Biochemistry 11, 1623--1629) was 4.5, which is higher by about one pH unit than the pK value determined by our group (Kuramitsu et al. (1974) J. Biochem. 76, 671--683; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170. We found that their pH difference titration data in the absence and presence of saccharides can be consistently interpreted in terms of our pK values of Asp 52, Glu 35, and Asp 101, if we assume that the pK value of another ionizable group (probably Asp 48) is perturbed on esterification of Asp 52.     

A possibility of a protein-bound water molecule as the ionizable group responsible for pKe at the alkaline side in human matrix metalloproteinase 7 activity

Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline pK(a) (pK(e1) and pK(e2)) values of about 4 and 10. The ionizable group for pK(e2) was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for pK(e2) [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940-1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. pK(e2) values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3-0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1-0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for pK(e2).     

Residual electrostatic effects in the unfolded state of the N-terminal domain of L9 can be attributed to nonspecific nonlocal charge-charge interactions

Residual electrostatic interactions in the unfolded state of the N-terminal domain of L9 (NTL9) were found by Kuhlman et al. [(1999) Biochemistry 38, 4896-4903]. These residual interactions are analyzed here by the Gaussian-chain model [Zhou, H.-X. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3569-3574]. The original model is made more realistic by replacing "standard" model-compound pK(a) values for ionizable groups by those measured by Kuhlman et al. in peptide fragments of NTL9. The predicted pH dependence of the unfolding free energy is in agreement with experiment over the pH range of 1-7 at ionic strengths of 100 and 750 mM. This indicates that the residual electrostatic effects in the unfolded state of NTL9 can be attributed to nonspecific nonlocal charge-charge interactions.     

Interaction of monodispersed and micellar phospholipids with an Agkistrodon halys blomhoffii phospholipase A2, in which the alpha-amino group had been modified to an alpha-keto group

The pH dependence of the binding constant of Ca2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the alpha-amino group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as alpha-amino group. The pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH-dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH-dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799-804), which was assigned as the alpha-amino group. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the intact and the alpha-NH2-modified enzymes was studied by the pH stat method at 25 degrees C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca2+. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C12PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the alpha-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Empirical parametrization of pK values for carboxylic acids in proteins using a genetic algorithm

Considerable effort has been devoted to the development of theoretical electrostatic methods to predict the pK values of ionizable residues in proteins. However, predictions appear often to be still at the qualitative or semi-quantitative level. We believe that, with the increasing number experimentally available pK values for proteins of known structure, an alternative approach becomes feasible: the empirical parametrization of the experimental protein pK database. Of course, in the long term, this empirical approach is no substitute for rigorous electrostatic analysis but, in the short term, it may prove to have useful predictive power and it may help to pinpoint the main structural determinants of pK values in proteins. Here we demonstrate the feasibility of the parametrization approach by fitting (using a genetic algorithm as fitting tool) the database for carboxylic acid pK values in proteins on the basis of an empirical equation that takes into account the two following kinds of effects: (1) long-range charge-charge interactions; (2) interactions of the given carboxylic acid group with its environment in the protein, which are described in terms of contributions from the different kind of atoms present in the protein (atomic contributions).     

Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential

The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context.     

Application of the Gaussian dielectric boundary in Zap to the prediction of protein pKa values

The results of two rounds of blind pK(a) predictions for ionizable residues in staphylococcal nuclease using OpenEye's legacy protein pK(a) prediction program based on the Zap Poisson-Boltzmann solver were submitted to the 2009 prediction challenge organized by the Protein pK(a) Cooperative and first round predictions were discussed at the corresponding June 2009 Telluride conference. To better understand these results, 21 additional sets of predictions were performed with the same program, varying the internal dielectric, reference pK(a), partial charge set, and dielectric boundary. The internal dielectric (ε(p)) and dielectric boundary were the two most important factors contributing to the quality of the predictions. Although the lowest overall errors were observed with a molecular dielectric boundary at ε(p) = 8, predictions using a smooth Gaussian dielectric boundary performed almost as well at lower ε(p) values because the Gaussian boundary implicitly accounts for a significant level of solvent penetration. Improved pK(a) predictions with the Gaussian boundary methodology will require better prediction and modeling of structural changes due to changes in ionization state, perhaps without resorting to the more exhaustive sampling of conformational states used by other recent continuum methods.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25 degrees C ionic strength 0.1 in the presence of 3-10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20).(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.     

pK values of the ionizable groups of proteins

We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25 degrees C are: 3.67+/-0.03 (alpha-carboxyl), 3.67+/-0.04 (Asp), 4.25+/-0.05 (Glu), 6.54+/-0.04 (His), 8.00+/-0.03 (alpha-amino), 8.55+/-0.03 (Cys), 9.84+/-0.11 (Tyr), and 10.40+/-0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N & T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N & T); His (6.54 this work vs. 6.3 N & T); alpha-amino (8.00 this work vs. 7.5 N & T); Cys (8.55 this work vs. 9.5 N & T); and Tyr (9.84 this work vs. 9.6 N & T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins.     

A statistical approach to the prediction of pK(a) values in proteins

We propose a simple model for the calculation of pK(a) values of ionizable residues in proteins. It is based on the premise that the pK(a) shift of ionizable residues is linearly correlated to the interaction between a particular residue and the local environment created by the surrounding residues. Despite its simplicity, the model displays good prediction performance. Under the sixfold cross test prediction over a data set of 405 experimental pK(a) values in 73 protein chains with known structures, the root-mean-square deviation (RMSD) between the experimental and calculated pK(a) was found to be 0.77. The accuracy of this model increases with increasing size of the data set: the RMSD is 0.609 for glutamate (the largest data set with 141 sites) and approximately 1 pH unit for lysine, with a data set containing 45 sites.     

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.     

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7)

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK(a) of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25-45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ?H°, of deprotonation were calculated. pK(e1) and pK(e2) of MMP-7 at 15-45°C were determined in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), from which ?H(o) for pK(e1) and pK(e2) was calculated. The ?H(o) for pK(e1) (-20.6±6.1kJmol(-1)) was similar to that for L-Glu (-23.6±5.8kJmol(-1)), and the ?H(o) for pK(e2) (89.9±4.0kJmol(-1)) was similar to those for L-Arg (87.6±5.5kJmol(-1)) and L-Lys (70.4±4.4kJmol(-1)). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK(e1). On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK(e2).     

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

Multiple nanosecond duration molecular dynamics simulations on the pore-lining M2 helix of the nicotinic acetylcholine receptor reveal how its structure and dynamics change as a function of environment. In water, the M2 helix partially unfolds to form a molecular hinge in the vicinity of a central Leu residue that has been implicated in the mechanism of ion channel gating. In a phospholipid bilayer, either as a single transmembrane helix, or as part of a pentameric helix bundle, the M2 helix shows less flexibility, but still exhibits a kink in the vicinity of the central Leu. The single M2 helix tilts relative to the bilayer normal by 12 degrees, in agreement with recent solid state NMR data (Opella et al., Nat Struct Biol 6:374-379, 1999). The pentameric helix bundle, a model for the pore domain of the nicotinic receptor and for channels formed by M2 peptides in a bilayer, is remarkably stable over a 2-ns MD simulation in a bilayer, provided one adjusts the pK(A)s of ionizable residues to their calculated values (when taking their environment into account) before starting the simulation. The resultant transbilayer pore shows fluctuations at either mouth which transiently close the channel. Proteins 2000;39:47-55.