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.     

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.     

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.     

Evidence for histidine in the active site of papain

Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-¹⁴C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.     

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle

F?rster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.     

Spin-labeling study on the depth of the active site of papain

Papain is alkylated with a series of haloacetamide spin labels with varying distance between the haloacetamide residue and the nitroxide portion. The dependence of τ_c on the distance and temperature revealed that the active site has a narrow neck and one auxiliary binding site around the neck. The depth of the active site is estimated about 10 A.     

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.     

Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate). Thus, in this sense the stabilization of the thioimidate does not reflect a similarity in structure between the bound thioimidate and the transition state.     

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.     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Definition of the site of reactivity of the ancestral protease of the papain type

Digestions of oxidized insulin B chain by the sulfhydryl proteases papain, chymopapain, papaya peptidase A, actinidin, bromelain, ficin and asclepains     

Yeast thioltransferase--the active site cysteines display differential reactivity

Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae. The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography. Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography. The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system. Both proteins showed similar amino acid compositions and identical N-termini. The major peak of thioltransferase was extensively characterized. Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics. The enzyme activity had a pH optimum of 9.1. The protein has 106 amino acid residues with two cysteines and no arginine. The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin. The two cysteines at the active site displayed different reactivities to iodoacetamide. Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0. The enzyme was completely inactivated when the Cys26 was carboxymethylated. A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity. The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH.     

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.     

A novel reversible thiol-specific spin label: papain active site labeling and inhibition

A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5–7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.