1H-NMR study on the tautomerism of the imidazole ring of histidine residues. II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease A

The NMR titration curves of proton chemical shifts were observed for the C2 protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250-340 nm region of circular dichoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N1-H tautomer of His-12, more than 8 for the N3-H tautomer of His-12, 7.0 for the N1-H tautomer of His-119, and 6.4 for the N3-H tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

Ionization constants and thermodynamic parameters of histidine and derivatives

The pK_a values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pK_a values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pK_a in histidine and its studied derivatives. The N^τ-H tautomer is favored over the N^π-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.     

Comparative proton NMR studies of bovine semen and pancreas ribonucleases

The fine structure of bovine semen RNAase was studied with proton NMR spectroscopy making use of the four-protein system constituted by dimeric bovine semen RNAase, its catalytically active monomeric bis-(S-carboxymethyl-31,32) derivative, the naturally monomeric RNAase A from the pancrease of the same species, and dimerized RNAase A. Only four histidine C-2 H resonances were observed in the aromatic spectrum of bovine semen RNAase, which belong to the four histidine residues present in the sequence of bovine semen RNAase subunits at positions identical with those of the histidines of RNAase A. This is indicative of identical environments for the individual histidine residues in both subunits. These resonances were assigned (i) by comparing their titration curves with the corresponding curves obtained with RNAase A and with monomeric bovine semen RNAase and (ii) by evaluating the effects on their titration curves of nucleotide binding. Very similar NMR parameters were measured for His-105 and also for His-119 of seminal and pancreatic RNAase, while His-12 was found to have different environments in the two proteins. The distinctive NMR features of His-48 in bovine semen RNAase confirmed the role of the hinge regions of the subunits in maintaining the dimeric structure of the protein. While monomerization of the seminal enzyme reduced the differences between the histidine C-2 H resonances of RNAase A and bovine semen RNAase, dimerization of RNAase A did not affect the NMR spectrum of this protein, thus indicating as unlikely the possibility that the quaternary structure of bovine semen RNAase resembles that of dimerized RNAase A.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the NMR spectrum of ribonuclease A in H2O.

Described herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK_a of 6.1 in H₂O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pK_a's and constant area over the above pH range in His-119-N₁-carboxymethylated-RNase A and des-(121–124)-RNase A, thus eliminating the imidazole N₃ proton in the His 119-Asp 121 hydrogen bond. In His-12-N₁-carboxymethylated-RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N₁ proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 45 in the crystal.     

Catalytic activity of Ntau-carboxymethylhistidine-12 ribonuclease: pH dependence.

The pH-dependence of RNAase A and of Ntau-carboxymethylhistidine-12-RNAase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase) catalysis was studied. Apparent acid dissociation constants were obtained by least squares analysis of the kinetics data. These dissociation constants were compared with pKa values of model imidazole compounds, and with pKa values of histidine residues 12 and 119 on the protein. The shapes of the kcat versus pH profiles for RNAase A and its carboxymethyl derivative are very similar, from which it is concluded that the mechanism of catalysis is closely similar in the two proteins. Apparent pKa values obtained from the kinetic data are higher for the carboxymethylated protein than for RNAase A, as are the pKa values of residues 12 and 119. The similar shifts are consistent with the conclusions that both these residues are functionally significant in native and modified enzyme, and that an unblocked tau-nitrogen on histidine-12 is not essential for activity. From the enzyme's catalytic dependence on pH, and the NMR determined pKa values we propose that histidine 12 and 119 function catalytically in their basic and acidic forms respectively.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

Proton nuclear magnetic resonance studies of histidine residues in rat and other rodent pancreatic ribonucleases. Effects of pH and inhibitors

Proton nuclear magnetic resonance spectra of the histidine residues in bovine and rat ribonuclease have been compared. The changes in chemical shift on titration and on binding of cytidine-3′-monophosphate and cytidine-2′-monophosphate have been followed. In the presence of the cytidine derivatives the spectra of both enzymes resemble each other more than those of the free enzymes. With these inhibitors, two histidines in rat ribonuclease exhibit the same pK values and shifts as the active site residues histidine 12 and 119 in the bovine enzyme. Their pK values in the inhibitor-free rat enzyme are about 0.4 higher than in the beef enzyme, which can be explained by the substitution at the entrance of the active site cleft of arginine 39 in the beef enzyme by serine in the rat enzyme. Rat ribonuclease contains one histidine with a rather high pK value of 7.6. The cytidine derivatives affect its chemical shift in exactly the same way as the shift of histidine 48 in bovine ribonuclease. The high pK value of this residue in rat ribonuclease can be explained by assuming a strong hydrogen bridge with glutamic acid 16. The other two histidines in rat ribonuclease have rather low pK values of 6.1 and 6.3. The histidine with a pK value of 6.3 has been assigned to position 105 and that with a pK value of 6.1 to position 73.The closer resemblance of the active sites of bovine and rat ribonuclease in the presence of inhibitors than in the inhibitor-free enzymes makes the concept of induced fit interesting from an evolutionary point of view.The characteristic downfield shift of the protonated form of histidine 119 in the complexes of bovine and rat ribonuclease with cytidine-3′-monophosphate is not observed with uridine-3′-monophosphate, suggesting non-identical binding of these pyrimidine nucleotides.Some preliminary results on the nuclear magnetic resonance properties of the histidine residues in coypu and chinchilla pancreatic ribonuclease have been obtained.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.     

Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique.

Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Nucler magnetic resonance studies of the unfolding of pancreatic ribonuclease.

The thermal unfolding of ribonuclease at a number of pH values has been studied by ¹H nuclear magnetic resonance spectroscopy, under conditions where the unfolding is fully reversible and concentration-independent. At pH¹ 5.5 (uncorrected for the deuterium isotope effect) there is evidence for a conformational change affecting His-48 and perhaps a methionine residue at temperatures below the major thermal transition. No evidence for intermediates in the major transition was found. The product of thermal unfolding under these conditions is not a random coil, and the remaining elements of structure probably include a phenylalanine and two histidine residues. At pH¹ 1.5 and pH¹ 2.9, the product of thermal unfolding is closer to a random coil, and under these conditions the changes in area of the histidine C₍₂₎H resonances with temperature give evidence for the existence of an intermediate in the unfolding process in which His-12 and His-119 are in a solvent-like environment, while His-48 and His-105 are not (see Westmoreland &; Matthews (1973)). The changes in the spectra of ribonuelease between pH¹ 5.5 and pH¹ 1.5 are described, and the possible relation between these changes and the alterations in thermal unfolding with pH are discussed.     

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration

The dissociation constants (pK_ms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a ¹³C nuclear magnetic resonance (NMR) titration method employing their N-¹³CH₃ (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 μM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pK_m values by allowing that the phenothiazines in the aqueous phase could be neglected. The pK_m values were calculated from the chemical shift dependence of the N-dimethyl ¹³C NMR signal on the pH value of sample solutions. The pK_m values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pK_m values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pK_m decrease and provided their precise values.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

Correlation of exchangeable (NH) and nonexchangeable (C2H) histidine resonances in the proton NMR spectrum of ribonuclease A in aqueous solution.

The ionization characteristics of the hydrogen-bonded His 12 N₁ proton observed to titrate between 11 to 13 ppm in the nmr spectrum of ribonuclease A in H₂O solution are compared with the ionization characteristics of the four histidine C₂ protons in the enzyme. Comparison of the pK_a's of the enzyme in H₂O and D₂O in the absence and presence of cytidine monophosphate (?5′, ?3′, and ?2′) inhibitors, line widths in the presence of Cu II at pH 3.6 and 5.6, and chemical shifts in the presence of AgNO₃ permit a correlation of the exchangeable His 12 N₁ proton with the active site histidine C₂ proton exhibiting the lower ionization pK_a. The histidines with pK_a of 5.1 and 5.6 in ribonuclease A in the absence of salt are assigned in this study to His 12 and His 119, respectively.     

The structure of carbonyl horseradish peroxidase: spectroscopic and kinetic characterization of the carbon monoxide complexes of His-42 → Leu and Arg-38 → Leu mutants

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pK _as at 7.4 and 4.0. Data obtained with the His-42?→?Leu [(H42L)HRPC*)] and Arg-38?→?Leu [(R38L)HRPC*] mutants allowed the pK _a at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν?(CO) bands at 1934?cm^–1 and 1905?cm^–1 whose relative intensity changed with pH, showing an acidic and a basic pK _a as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118?:?3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2?cm^–1 (pH?7.0) and 1941.5?cm^–1 (pH?5.0) respectively. Acidic and alkaline pK _as were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pK _a at ～pH?4.0 but no alkaline pK _a. (R38L)HRPC*-CO exhibited a single pK _a at pH?6.5. Shifts of 2–3?cm^–1 in ν?(CO) with (H42L)HRPC*-CO in D₂O show that a distal residue is H-bonding to the CO in this variant at both pD?7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν?(CO) band was observed at pD?5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH?8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pK _a between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.     

Histidines 345 and 378 of <Emphasis Type="Italic">Bacillus stearotheromophilus</Emphasis> leucine aminopeptidase II are essential for the catalytic activity of the enzyme

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the K_m value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in K_cat/K_m for H227L was partly caused by a 3.9-fold increase in K_m value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.