scholarly journals Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives

1986 ◽  
Vol 233 (1) ◽  
pp. 119-129 ◽  
Author(s):  
B S Baines ◽  
K Brocklehurst ◽  
P R Carey ◽  
M Jarvis ◽  
E Salih ◽  
...  
Keyword(s):  
Close Contact ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Resonance Raman ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Ph Values ◽  
Covalent Chromatography

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M−1 × cm −1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2′-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2′-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

scholarly journals Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4

1985 ◽  
Vol 227 (2) ◽  
pp. 511-519 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Thiol Group ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Active Centre ◽  
Ph Values ◽  
Cathepsin H ◽  
Covalent Chromatography

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2′-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2′-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2′-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.

scholarly journals Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II

1984 ◽  
Vol 223 (3) ◽  
pp. 649-657 ◽  
Author(s):  
K Brocklehurst ◽  
P R Carey ◽  
H H Lee ◽  
E Salih ◽  
A C Storer
Keyword(s):  
Steady State ◽  
Kinetic Study ◽  
Close Contact ◽  
Cysteine Proteinase ◽  
Resonance Raman ◽  
Kinetic Studies ◽  
Cysteine Residue ◽  
Catalytic Site ◽  
Steady State Kinetic ◽  
State Kinetic

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.

scholarly journals Differences in the interaction of the catalytic groups of the active centres of actinidin and papain Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes

1981 ◽  
Vol 197 (3) ◽  
pp. 739-746 ◽  
Author(s):  
K Brocklehurst ◽  
B S Baines ◽  
J P Malthouse
Keyword(s):  
Cysteine Proteinase ◽  
Thiol Group ◽  
Interactive System ◽  
Active Centre ◽  
Ph Values ◽  
Hydrophobic Binding ◽  
Rapid Purification ◽  
Active Centres ◽  
Covalent Chromatography

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.

scholarly journals Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain

1989 ◽  
Vol 258 (3) ◽  
pp. 755-764 ◽  
Author(s):  
D Kowlessur ◽  
C M Topham ◽  
E W Thomas ◽  
M O'Driscoll ◽  
W Templeton ◽  
...  
Keyword(s):  
Transition State ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Catalytic Site ◽  
Amide Bond ◽  
Compound I ◽  
Binding Interactions ◽  
Transition State Geometry ◽  
S2 Subsite

1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2′-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2′-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

scholarly journals Differences in the chemical and catalytic characteristics of two crystallographically ‘identical’ enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment

1987 ◽  
Vol 247 (1) ◽  
pp. 181-193 ◽  
Author(s):  
E Salih ◽  
J P G Malthouse ◽  
D Kowlessur ◽  
M Jarvis ◽  
M O'Driscoll ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Thiol Group ◽  
Ion Pair ◽  
Low Ph ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Acid Media ◽  
Catalytic Sites

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2′-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2′-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2′-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2′-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups

1982 ◽  
Vol 205 (1) ◽  
pp. 205-211 ◽  
Author(s):  
B S Baines ◽  
K Brocklehurst
Keyword(s):  
Carica Papaya ◽  
Cysteine Proteinase ◽  
Thiol Group ◽  
Catalytic Site ◽  
Interactive System ◽  
Ionization State ◽  
Active Centre ◽  
Ph Values ◽  
Activity Loss

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely ‘essential’ for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2′-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.

scholarly journals Investigation of the catalytic site of actinidin by using benzofuroxan as a reactivity probe with selectivity for the thiolate-imidazolium ion-pair systems of cysteine proteinases. Evidence that the reaction of the ion-pair of actinidin (pKI 3.0, pKII 9.6) is modulated by the state of ionization of a group associated with a molecular pKa of 5.5

1983 ◽  
Vol 213 (3) ◽  
pp. 713-718 ◽  
Author(s):  
E Salih ◽  
K Brocklehurst
Keyword(s):  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Ion Pair ◽  
The State ◽  
Catalytic Site ◽  
Analogous Reaction ◽  
Decreasing Ph ◽  
Electrophilic Reactivity

Benzofuroxan reacts with the catalytic-site thiol group of actinidin (EC 3.4.22.14, the cysteine proteinase from Actinidia chinensis) to produce stoicheiometric amounts of the chromophoric reduction product, o-benzoquinone dioxime, and of a catalytically inactive derivative of actinidin that is devoid of thiol and that is assumed to contain, initially at least, the sulphenic acid of cysteine-25. A similar result applies also to papain (EC 3.4.22.2). The rate of o-benzoquinone dioxime formation is neither increased by inclusion of 2-mercaptoethanol or hydroxylamine in the reaction mixture nor decreased by changing the solvent from H2O to 2H2O. The change of solvent was shown to be without effect also on the rate of reaction of benzofuroxan with papain. These results suggest that the reactions of benzofuroxan with both actinidin and papain involve rate-determining attack of the catalytic-site thiol group to produce an intermediate adduct that then reacts rapidly with water to form enzyme sulphenic acid and o-benzoquinone dioxime. The pH-dependence of the second-order rate constant for the reaction of benzofuroxan with actinidin was determined in the pH range 4.3-10.2. In marked contrast with the analogous reaction of papain (reported by Shipton & Brocklehurst [(1977) Biochem. J. 167, 799-810]) the pH-k profile for the actinidin reaction clearly contains a sigmoidal component with pKa 5.5, in which k increases with decreasing pH. These data together with the molecular pKa values for S-/ImH+ ion-pair formation and decomposition (3.0 and 9.6) suggest that the combined nucleophilic-electrophilic reactivity of the ion-pair of actinidin might be controlled by the state of ionization of another ionizing group, associated with the molecular pKa of 5.5. The pH-dependence of k for the reaction of actinidin with benzofuroxan at 25 degrees C at I 0.1 in aqueous buffers containing 6.7% (v/v) ethanol is probably adequately described by: k = k1/(1 + [H+]/KI + KII/[H+]) + k2/(1 + [H+]/KII + KIII/ [H+] + k3/(1 + [H+]/KIII) in which kI = 2.55 M -1 X s -1, k2 = 1.35 M -1, k3 = 0.93 M -1 X s -1, pKI = 3.0, pKII = 5.5 and pKIII = 9.6. By contrast, the analogous reaction of papain may be described by the same equation but with kI = 0, k2 = 2.2 M -1 X s -1, k3 = 1.3 M -1 X s -1, pKII = 3.6 and pKIII = 9.0.

scholarly journals Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites

1986 ◽  
Vol 238 (1) ◽  
pp. 103-107 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Ion Pair ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Weakly Alkaline ◽  
Stem Bromelain ◽  
Ph Range

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.

The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

scholarly journals A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition

1990 ◽  
Vol 266 (3) ◽  
pp. 645-651 ◽  
Author(s):  
W Templeton ◽  
D Kowlessur ◽  
E W Thomas ◽  
C M Topham ◽  
K Brocklehurst
Keyword(s):  
Transition State ◽  
Binding Site ◽  
Kinetic Data ◽  
Model Building ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Structural Basis ◽  
Compound I

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

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