Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Br?nsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.     

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.     

Active-site ionizations of papain. An evaluation of the potentiometric difference titration method

The underlying assumption of the potentiometric difference titration method, as applied to the evaluation of the sulfhydryl-dependent ionizations in the active site of papain, is that the pKa of His-159 is independent of whether the neighboring sulfhydryl (Cys-25) is protonated or methylthiolated. That this idealized assumption may not strictly apply is indirectly indicated by the larger pKa of His-159 in S-methylpapain versus that in S-methylthiopapain, as determined from fluorometric titrations (delta pKa = 0.32 +/- 0.05, 25 degrees C). On the basis of the Wegscheider principle of the equivalence of protons and methyl groups, the potentiometric difference titration method will underestimate the concentration of thiolate-imidazolium ion pair in the active site versus that of the thiol-imidazole tautomer, provided that there is no significant H-bonding interaction in the latter species.     

Proton equilibria in the binding of Zn2+ and of methylmercuric iodide to papain.

The proton liberation on the binding of zinc chloride and methylmercuric iodide to the (essential) thiol group of papain has been examined as a function of pH. This was carried out by (a) direct titration of the protons on the addition of the metal compound to active papain and (b) measurement of the extent of inhibition of enzyme activity by the metal compound as a function of pH. It was found that in the neutral pH range the thiol group or the neighbouring imidazole group in the free enzyme carries one proton, at low pH both groups do so, whereas at high pH neither group carries a proton. The pK values of the free enzyme that govern the proton release, 4.2 and 8.5, correspond to those that govern overall activity. Both from the experiments with methylmercuric iodide and from fluorescence measurements of methylmercuric papain, it was established that the imidazole group in the latter compound exhibits a pK of 5.4. Taking recent data into account, it was considered that the ion pair of thiolate anion and imidazolium cation, proposed by Polgar, is the best approximation to describe the charge distribution in the active centre and to explain the reaction mechanism.     

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl-l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S_N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl-l-phenylalanyl-l-valyl-l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl-l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).     

Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system.

1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.     

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.     

The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.     

Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase.

The reactivity and the mode of activation of the essential--SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1 equals 5.5, pK2 equals 8.2). It is concluded that there are two reactive forms of the--SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK2 equals 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 equals 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.     

Investigation of the active site of papain with fluorescent probes

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) and 7-(2'-hydroxyethylthio)-NBD (obtained from NBD chloride and mercaptoethanol) undergo a reversible spectral change in alkaline solution that depends respectively on a single apparent pK(a) 9.76 (at 25 degrees C) and 8.81 (at 32 degrees C). In acid solution however no spectral change was observed. NBD chloride reacts slowly with papain at pH7, but the rate of inhibition increases at lower pH and depends on an apparent pK(a) of 3.7 (at 35 degrees C), which has been tentatively assigned to the carboxyl group of aspartic acid-158. The spectral properties of NBD-papain indicate that the thiol group of cysteine-25 is the site of reaction. The intensity of the fluorescence-emission spectrum of NBD-papain depends on a single pK(a) of 4.2 (at 26.7 degrees C). The intensity of the fluorescence-emission spectrum of the mixed disulphide formed from papain and 7-(2'-mercaptoethylamino)-NBD (obtained from NBD chloride and cysteamine) depended on a single pK(a) of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27 degrees C). This small change to lower pK(a) value in a medium of lower dielectric constant is characteristic of a cationic acid. The only acid of this type in the active-site region is the conjugate acid of histidine-159.     

Deciphering the role of histidine 252 in mycobacterial adenosine 5'-phosphosulfate (APS) reductase catalysis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5'-phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pK(a) of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.     

Identification of the functional ionic groups of papain by pH/rate profile analysis.

The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.     

Refined x-ray structure of papain.E-64-c complex at 2.1-A resolution.

E-64-c, a synthetic cysteine protease inhibitor designed from E-64, binds to papain through a thioether covalent bond. The x-ray diffraction data for 2.1-A resolution were used to determine the three-dimensional structure of this complex and refined it to R = 0.159. 0.159. In the complex structure, the configurational conversion from S to R took place on the epoxy carbon of E-64-c, implying that the nucleophilic attack of the Cys-25 thiol group occurs at the opposite side of the epoxy oxygen atom. The leucyl and isoamylamide groups of E-64-c were strongly fixed to papain S subsites by specific interactions, including hydrogen bonding to the Gly-66 residue. The carboxyl-terminal anion of E-64-c formed an electrostatic interaction with the protonated His-159 imidazole ring (O-...HN+ = 3.76 A) and consequently prevented the participation of this residue in the hydrolytic charge-relay system. No significant distortion caused by the binding of E-64-c was shown in the secondary structure of papain. It is important to note that inhibitor and substrate have opposite binding modes for the peptide groups. The possible relationship between the binding mode and inhibitory activity is discussed on the basis of the crystal structure of this complex.     

Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.     

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)     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Interaction of ADP with skeletal and cardiac myosin and their active fragments observed by proton release.

The technique of proton release measurement has been used to explore the binding of ADP to skeletal and cardiac myosins and their active fragments in a variety of conditions. It has proved possible to obtain binding profiles on intact myosin in the filamentous, undissolved form in physiological solvent conditions. Binding constants are given. At higher ionic strength (0.5 M potassium chloride) the binding profile of magnesium-ADP. is compatible with the presence of two types of site, differing from one another both in respect of affinity and the number of protons released per site. Studies with cardiac myosin reveal no such indications of heterogeneity, and are consistent with the presence of a single population of thermodynamically indistinguishable sites. In the absence of divalent cations, in solutions containing potassium ions and EDTA, ADP binds with absorption rather than liberation of protons. The pH profile of proton absorption at saturation can be fitted in terms of an ionising group with an unperturbed pK of 9.4, and at least one of lower pK(5.9). The dissociation constant (pH8 at 5 degrees C) is about 8 microM, and the affinity for uncomplexed ADP is thus only slightly weaker than that for magnesium-ADP     

Contribution of histidine residues to the conformational stability of ribonuclease T1 and mutant Glu-58----Ala

The pK values of the histidine residues in ribonuclease T1 (RNase T1) are unusually high: 7.8 (His-92), 7.9 (His-40), and 7.3 (His-27) [Inagaki et al. (1981) J. Biochem. 89, 1185-1195]. In the RNase T1 mutant Glu-58----Ala, the first two pK values are reduced to 7.4 (His-92) and 7.1 (His-40). These lower pKs were expected since His-92 (5.5 A) and His-40 (3.7 A) are in close proximity to Glu-58 at the active site. The conformational stability of RNase T1 increases by over 4 kcal/mol between pH 9 and 5, and this can be entirely accounted for by the greater affinity for protons by the His residues in the folded protein (average pK = 7.6) than in the unfolded protein (pk approximately 6.6). Thus, almost half of the net conformational stability of RNase T1 results from a difference between the pK values of the histidine residues in the folded and unfolded conformations. In the Glu-58----Ala mutant, the increase in stability between pH 9 and 5 is halved (approximately 2 kcal/mol), as expected on the basis of the lower pK values for the His residues in the folded protein (average pK = 7.1). As a consequence, RNase T1 is more stable than the mutant below pH 7.5, and less stable above pH 7.5. These results emphasize the importance of measuring the conformational stability as a function of pH when comparing proteins differing in structure.     

A kinetic and fluorimetric investigation of papain modified at tryptophan-69 and -177 by N-bromosuccinimide

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan-69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pK(a) of the acidic limb of the pH profile by more than 1 pH unit for both k(cat.) and k(cat.)/K(m). The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan-69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan-177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pK(a) of 4.2. Histidine-159, which is in close contact with tryptophan-177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan-177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pK(a) determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N-benzyloxycarbonylglycylglycine amide and N-benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan-177 were shown to be structural features of the S(2) and S(1)' subsites respectively.     

Interaction of transition metal ions with ribonuclease A. II. The selective effects of Mn2+, Zn2+, Cd2+ and Hg2+ on the histidine magnetic resonance.

Zn2+, Cd2+ and Hg2+ inhibit ribonuclease but Mn2+ does not except at very high concentrations. By high resolution NMR one can detect in the pH range 5-8 the C-2 protons of histidines 105, 12, and 119. The inhibiting ions produce large shifts of the resonance of His-12 but not of His-105. On the other hand Mn2+ broadens the C-2 proton of His-105 much more than it does those of His-12 and 119. The selective shifts suggest that the mechanism of inhibition is binding at or near the active site of which His-12 and 119 are a part. The selective broadening is a consequence of binding of the Mn2+ to a site very far from the active site but closer to His-105.