Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups.

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.     

Ionization of clusters. IV. Cooperativity and allosterism derived from shared clusters in macromolecules.

Clusters of ionizable groups are examined for conditions that develop cooperativity (1) on the binding of protons, and (2) on the binding of an associated ligand when the clusters are shared between domains or subunits in macromolecules. Cooperative binding isotherms for protons have long been observed (but not emphasized as cooperative binding) when studies have been done on clusters for the evaluation of metal ion complexation [A. E. Martell & M. Calvin (1952) Chemistry of the Metal Chelate Compounds, Prentice-Hall, Englewood Cliffs, New Jersey]. Reactions are formulated in this paper to show that anions, chelating to positively charged clusters, are also capable of developing the cooperative binding of protons. Extension of these simple reactions to those where clusters of ionizable groups are shared between domains of macromolecules provides models for cooperative binding, which include the allosteric, Bohr, anion, and cation effects in proteins.     

Studies on the N-acetyl-beta-D-hexosaminidase B from germinating Lupinus luteus L. seeds. II. Mechanism and inhibition with some 2-acetamido-2-deoxyaldono(1----4)lactones

The N-acetyl-beta-D-hexosaminidase B of germinating yellow lupin seeds catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and -galactosaminide substrates. The investigation of the pH dependence of the kinetic parameters (Vmax and Vmax/Km) demonstrated that two common ionizable groups (probably two carboxyl groups) play an essential role in the catalysis. That is, the enzyme has a lysozyme-like splitting mechanism, and the possibility of an anchimeric assistance provided by the acetamido group seems to be negligible. The presence of a deprotonated carboxyl group near the glycosidic linkage was also supported by inhibition with 1-thio substrate analogues. On the other hand, some 2-acetamido-2-deoxyaldono(1----4)lactones proved to be effective inhibitors of the hexosaminidase with the exception of the D-arabinose derivative, which can be explained by high stereospecificity in the binding.     

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.     

pH dependence of the binding constant of a phospholipase A2 from Agkistrodon halys blomhoffii venom to micelles of n-hexadecylphosphorylcholine

The phospholipase A2 from the venom of A. halys blomhoffii was titrated with micellar n-hexadecylphosphorylcholine (an analog of lysolecithin) by following the tryptophyl fluorescence change 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 that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 10.0 and 6.7 for the apoenzyme and its Ca2+ complex, respectively. The binding constant of the enzyme to the substrate micelle was found to be enhanced by Ca2+ binding to the enzyme. 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 5.16 to 5.67 and from 6.45 to 6.6. The pH-dependence curve for the enzyme-Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of a single ionizable group from 5.55 to 5.76. The former ionizable group was assigned as Asp 49, to which Ca2+ ion can coordinate, and the latter as His 48 in the active site. No participation of the alpha-amino group with a pK value of 7.30 was observed. The binding constant of the enzyme to the substrate micelle, Kmic = 0.45-2.3 X 10(6) M-1, was found to be far greater than that to the monomeric substrate, Kmon = 0.2-1.0 X 10(4) M-1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.     

A kinetic study of the reaction of horseradish peroxidase with hydrogen peroxide.

A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound I of th;e enzyme. Analysis of the results, indicates that there are two kinetic influencing, ionizable groups on the enzyme with pKa values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H2O2. A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W.D. & Dunford, H.B.: Biochemistry 7, 2054-2062 (1968)) has been extended to down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pKa values of 2.9 and 3.9.     

Hydrogen ion effects of the vertebrate photoreceptor. The pK's of ionizable groups affecting cell permeability.

The permeability of the vertebrate photoreceptor was studied as a function of pH and other parameters. The permeability was measured as the rate of decay of a sequence of distal PIII responses from an isolated frog retina (Rana catesbeiana) after reestablishing the transmembrane sodium gradient in the presence of ouabain. The rate of decay was slowed at increasing stimulus light intensities, with increased extracellular calcium concentration (0.1 to 5 mm), and at extracellular pH's above 7.6 and below 6.6 at lower stimulus light intensities. The effects of stimulus intensity and calcium are consistent with previously published data which showed that these parameters reduced the sodium conductance of the vertebrate photoreceptor. The pH data indicate that two externally available ionizable groups with pK's of approximately 7.6 and below 6.6 are affecting the permeability of the photoreceptor cell.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.     

Computation of the dipole moments of proteins.

A simple and computationally feasible procedure for the calculation of net charges and dipole moments of proteins at arbitrary pH and salt conditions is described. The method is intended to provide data that may be compared to the results of transient electric dichroism experiments on protein solutions. The procedure consists of three major steps: (i) calculation of self energies and interaction energies for ionizable groups in the protein by using the finite-difference Poisson-Boltzmann method, (ii) determination of the position of the center of diffusion (to which the calculated dipole moment refers) and the extinction coefficient tensor for the protein, and (iii) generation of the equilibrium distribution of protonation states of the protein by a Monte Carlo procedure, from which mean and root-mean-square dipole moments and optical anisotropies are calculated. The procedure is applied to 12 proteins. It is shown that it gives hydrodynamic and electrical parameters for proteins in good agreement with experimental data.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Spatial structure of cro-repressor in a solution. II. Effect of ionized groups

The technique of 1H NMR spectroscopy and absorption UV spectroscopy were used to study the ionization of the tyrosine phenol cycles and the effect of ionizable groups on the chemical shifts of signals from protons in the side chains of several amino acid residues. The microenvironment of these residues was established by analysing the titration curves. The mutual orientation of two functionally important adjacent alpha-helical protein regions was determined in solution. The signals from methionine residues belonging to different regions of the secondary structure were assigned in the NMR spectrum. The results indicate that the spatial structure of the repressor is similar in solution an in the crystal. They confirm the model proposed for the cro repressor interaction with DNA and based on the data of X-ray diffraction analysis.     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Environmental effects on the protonation states of active site residues in bacteriorhodopsin.

Finite difference solutions of the Poisson-Boltzmann equation are used to calculate the pKa values of the functionally important ionizable groups in bacteriorhodopsin. There are strong charge-charge interactions between the residues in the binding site leading to the possibility of complex titration behavior. Structured water molecules, if they exist in the binding site, can have significant effects on the calculated pKa by strongly stabilizing ionized species. The ionization states of the Schiff base and Asp-85 are found to be strongly coupled. Small environmental changes, which might occur as a consequence of trans-cis isomerization, are capable of causing large shifts in the relative pKa values of these two groups. This provides an explanation for the protonation of Asp-85 and the deprotonation of the Schiff base in the M state of bacteriorhodopsin. The different behavior of Asp-85 and Asp-212 is discussed in this regard.     

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.     

pH dependency of kinetic parameters and reaction mechanism of beef liver catalase.

The pH-dependence of the kinetic parameters in H2O2 decomposition by beef liver catalase was investigated. At pH 7.0, the ternary complex (ESS) decomposition rate was about 100 times faster than ESS formation (42 microM H2O2), and the value of the Michaelis constant was 0.025 M. From ethanol competition experiments, two different proton dissociation constants of the enzyme (pKe1 = 5.0, pKes2 = 5.9) were obtained for the binding of first and second H2O2 molecules. Another pKa value (pKes1) of 4.2 was obtained from the pH dependence of overall rate constant (ko). The reaction mechanism of catalase was discussed in relation to these ionizable groups.     

On the environment of ionizable groups in globular proteins

The environment of ionizable groups in 36 proteins is characterized in terms of solvent-accessibility, salt-bridge formation and hydrogen-bonding. Possible implications of our results as to the protonation state of buried ionizable groups are considered and patterns useful for model building studies on proteins are derived. The most interesting finding is that there are on average two completely buried ionizable groups per protein of which at least 20% do not form saltbridges. However, all buried ionizable groups form hydrogen bonds with neutral polar groups.     

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)     

Development of an equation for potentiometric titration of polyelectrolytes using a periodic lattice model. A numerical analysis of interactions among ionizable groups

A numerical method is presented for analysing the potentiometric titration behavior of linear polyelectrolytes. A polyelectrolyte molecule is treated as a one-dimensional lattice containing a large number of lattice points, each of which has an identical ionizable group. In this method, the polyelectrolyte model lattice is divided into identical repeating unit cell systems with a finite number of ionizable groups to calculate the thermodynamic partition function of the system of polyelectrolyte solution. The electrostatic interaction between ionized groups adopted in the present study is given by the Debye-Hückel type screened Coulombic potential. The titration behavior as well as several thermodynamic quantities is derived by making a canonical ensemble summation of all states in a unit cell system under an appropriate periodic boundary condition. This method serves as a model of the two-step ionization often appearing in polyions with strong neighboring interactions such as homo- and copolymers of maleic acid. Several characteristics of the titration behavior of these polyelectrolytes are well reproduced by using a lattice model with pairwise intervals, including the effects of hydrogen bond formation and change in dielectric constant of the medium around an ionizable group. In addition, this method is valid for a more detailed analysis of the titration behavior of polyelectrolytes with various kinds of arrangements of charged groups.     

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.