Potentiometric determination of ionizations at the active site of papain.

The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.     

QM/MM study of the active site of free papain and of the NMA-papain complex.

Hybrid quantum mechanical/molecular mechanical (QM/MM) calculations using restricted and unrestricted Hartree-Fock and B3LYP ab initio (QM) and Amber force field (MM), respectively, have been applied to study the catalytic site of papain in both free and substrate bonded forms. Ab initio geometry optimizations have been performed for the active site of papain and the N-methyl-acetamide (NMA)-papain complex within the molecular mechanical treatment of the protein environment. A covalent tetrahedral intermediate structure could be obtained only when the amide N atom of the substrate molecule was protonated through a proton transfer from the His-159 in the catalytic site. Our results support the previous assumption that a proton transfer from His-159 to the amide N atom of the substrate occurs prior to or concerted with the nucleophilic attack of the Cys-25 sulfur atom to the carbonyl group of the substrate. The electron correlation effect will reduce the proton transfer barrier. Therefore, this proton transfer can be easily observed in the B3LYP/6-31G* calculations. The HF/6-31G* method overestimates the reaction barrier against this proton transfer. The sulfur atom of Cys-25 and the imidazole ring of His-159 are found to be coplanar in the free form of the enzyme. However, the rotation of the imidazole ring of His-159 was observed during the formation of the tetrahedral intermediate. Without the papain environment, the coplanar thiolate-imidazolium ion pair RS-...ImH+ is much less stable than the neutral form of RSH....Im. Within the protein environment, however, the thiolate-imidazolium ion pair becomes more stable than its neutral form by 4.1 and 0.4 kcal/mol in HF/6-31G* and B3LYP/6-31G* calculations, respectively. The barrier of proton transfer from S-H group of Cys-25 to the imidazole ring of His-159 was reduced from 22.0 kcal/mol to 15.2 kcal/mol by the protein environment in HF/6-31G* calculations. This barrier is found to be much smaller (2.5 kcal/mol) in B3LYP/6-31G* calculations.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pKa of 8.4 +/- 0.1. The simplest interpretation is that the apparent pKa is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0. 0213 at 25 degrees C, corresponding to DeltaG'o = +2.3 kcal mol-1. From this value and the standard free energy change of -10.9 kcal mol-1 for the hydrolysis of ATP to AMP and PPi, we calculate that DeltaG'o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol-1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pKa of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the alpha,beta-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues. The spectrum is dominated by the contribution of Trp-35. The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75. This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989). This histidine residue is close to Trp-94. The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction. The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein. Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94.     

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: comparison of A and B isozymes

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-l-serine by a thiol to give l-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 A and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 A from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an approximately 10-fold lower Km for O-acetyl-l-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the alpha-aminoacrylate intermediate at any concentration of O-acetyl-l-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the alpha-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the alpha-amine's pKa. The pH dependence of the first-order rate constant for decay of the alpha-aminoacrylate intermediate to give pyruvate and ammonia gives a pKa of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with l-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the alpha-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.     

Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy

A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.     

Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional 1H NMR.

A series of two-dimensional (2D) correlated 1H NMR spectra of reduced and oxidized Escherichia coli thioredoxin have been used to probe the effects of pH in the vicinity of the active site, -Cys32-Gly-Pro-Cys35-, using the complete proton resonance assignments available for thioredoxin. In either oxidation state, the majority of residues of the thioredoxin molecule remain unchanged between pH 5.7 and pH 10, as indicated by the identical chemical shifts of the C alpha H, C beta H, and other protons. In reduced thioredoxin, a fairly widespread region around the active-site dithiol is affected by the titration of a group or groups with pKa approximately 7.1-7.4 in 2H2O. Another titration, with pKa approximately 8.4, affects a smaller region of the protein. Oxidized thioredoxin contains a disulfide and no free thiol groups; nevertheless, the proton resonances of many groups in the active-site region were observed to titrate with a pKa of 7.5, probably as a result of an abnormally high pKa value for the carboxyl group of the buried Asp-26 residue. For reduced thioredoxin, the results indicate that Asp-26 is titrating in this pH range, as well as both thiol groups. The new results are strongly suggestive that the mechanism of thioredoxin-catalyzed protein disulfide reduction may be critically dependent on proton transfer as well as electron transfer within the active site.     

13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.     

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Br?nsted relation. With reference to this Br?nsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.     

Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues

The properties of the six histidine residues of apocytochrome b5 have been investigated by using one- and two-dimensional proton NMR spectroscopy in order to probe the structure remaining after heme removal. Spectral assignments were arrived at by analyzing proton NOE connectivities, comparing them to those observed in the holoprotein, and inspecting the X-ray structure of the latter species. Each histidine residue was studied for its pKa value, interaction with the relaxation agent copper nitrilotriacetic acid, and reactivity toward bromoacetic acid. The four histidines which are not coordinated to the iron atom in the holoprotein (His-15, -26, -27, and -80) display in the major conformer of the apoprotein the same characteristic properties as in the holoprotein. Three of them are involved in specific interactions with the rest of the structure: His-15 and His-80 participate in hydrogen bonds, and His-27 is influenced by the nearby C-terminal segment. His-26 is the most exposed to the solvent. His-63 and His-39, which are located in the heme binding site, have distinct pKa values; they are affected differently by the copper agent and exhibit comparable reactivity toward bromoacetic acid, albeit milder than that of His-26. The results show that the heme binding residues are clearly distinguishable by their physicochemical properties and that several elements of native holoprotein structure are in place in the apoprotein. It is proposed that the structural influence of the heme is localized and that the amino- and carboxy-terminal segments form a structural unit providing stability to the apoprotein and supporting a fluctuating, partially folded binding site.     

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.     

Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility.

A new method for including local conformational flexibility in calculations of the hydrogen ion titration of proteins using macroscopic electrostatic models is presented. Intrinsic pKa values and electrostatic interactions between titrating sites are calculated from an ensemble of conformers in which the positions of titrating side chains are systematically varied. The method is applied to the Asp, Glu, and Tyr residues of hen lysozyme. The effects of different minimization and/or sampling protocols for both single-conformer and multi-conformer calculations are studied. For single-conformer calculations it is found that the results are sensitive to the choice of all-hydrogen versus polar-hydrogen-only atomic models and to the minimization protocol chosen. The best overall agreement of single-conformer calculations with experiment is obtained with an all-hydrogen model and either a two-step minimization process or minimization using a high dielectric constant. Multi-conformational calculations give significantly improved agreement with experiment, slightly smaller shifts between model compound pKa values and calculated intrinsic pKa values, and reduced sensitivity of the intrinsic pKa calculations to the initial details of the structure compared to single-conformer calculations. The extent of these improvements depends on the type of minimization used during the generation of conformers, with more extensive minimization giving greater improvements. The ordering of the titrations of the active-site residues, Glu-35 and Asp-52, is particularly sensitive to the minimization and sampling protocols used. The balance of strong site-site interactions in the active site suggests a need for including site-site conformational correlations.     

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.     

Zinc and cadmium 5-aminolevulinate dehydratase. Metal-dependent pH profiles

Native 5-aminolevulinic acid dehydratase contains zinc ions, which are essential for the enzymatic activity. Replacement of zinc by cadmium yielded an active enzyme whose kinetic parameters (kkat and Km) are similar to those of the zinc enzyme in the neutral pH range. However, the pH profiles of kcat and Km were different due to different pKa values. Two groups both with pKa values of 6.5 in the free zinc enzyme, but with pKa values of 7.0 in the cadmium enzyme were calculated from plots of log (kcat/Km) versus pH. On the other hand, the enzyme-substrate complex is controlled by one acidic group (zinc pKa = 6.0, cadmium pKa = 6.4) and one basis group (zinc pKa = 8.2, cadmium pKa = 7.7) as calculated from plots of log kcat versus pH. The Arrhenius plots for kcat of the two enzymes show no significant difference, the free energies of activation are 77.1 kJ/mol for the zinc and 76.8 kJ/mol for the cadmium enzyme. From this and from previous work it is concluded that the metal ions are located near the active site and influence the ionisations of essential amino acid residues. From the pH profiles of the modifying reaction and inhibition by diethylpyrocarbonate a histidinyl residue is inferred as one of the ionisable groups of the active site.     

pKa calculations for class A beta-lactamases: methodological and mechanistic implications.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.     

Proton nuclear magnetic resonance characterization of phospholipase A2 from Laticauda semifasciata

The molecular properties of phospholipases (PLases) A2 I and A2 III from a sea snake, Laticauda semifasciata, have been characterized by gel-filtration, as well as proton NMR, CD, UV absorption, and fluorescence spectroscopic methods. PLase A2 I exists as a monomer in aqueous solution in the presence or in the absence of Ca2+. The dissociation constants of the Ca2+-enzyme complexes have been determined for the two enzymes. The 270-mHz proton NMR spectra of PLases A2 I and A2 III have been measured, and the aromatic proton resonances of His-21 and His-48 in the active site have been assigned. By analyzing the pH dependence of the chemical shifts of the histidine proton resonances, pKa values have been determined for His-21 and His-48 with and without Ca2+. The conformational transitions have been found to take place at low pH or at high temperature (at approximately 65 degrees C). Fluorescence change of PLase A2 I upon addition of substrate analogs suggests that Trp-70 in PLase A2 I is involved in the binding to micellar substrates. The lack of Trp-70 in PLase A2 III is probably related to the low enzymatic activity as compared with that of PLase A2 I.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.