Keith Brocklehurst

[24] Two-protonic-state electrophiles as probes of enzyme mechanism

Publisher Summary A two-protonic-state electrophile is a compound that exhibits different electrophilic reactivities in each of two protonic or ionization states and can be used as reactivity probes with special characteristics that permit the detection and characterization of interaction in molecules in general and in enzyme active centers. In particular, it is used as reporter-group delivery vehicles and in the isolation of thiol-enzymes by covalent chromatography. This chapter presents the theoretical basis for the use as reactivity probes of two-protonic-state electrophiles in general and reviews the applications of pyridyl disulfides in the isolation and study of enzymes. Disulfides containing the 2-pyridyl moiety have been shown to be exceedingly versatile and valuable reagents for the isolation and study by both structural and kinetic methods of thiol-containing molecules. Covalent chromatography is a rapid isolation technique for thiol-enzymes and peptides, and provides considerable economies in both time and materials. It is reported that to draw mechanistic conclusions from kinetic data obtained by using reactivity probes, it is important to take account of the possibility that reactions of enzyme nucleophiles with site-specific reagents that are not formally substrate analogs might well proceed through the intermediacy of adsorptive complexes, albeit characterized by rather large dissociation constants.

Chapter 2 Cysteine proteinases

Publisher Summary The classification of proteinases is based on their possession of analogous mechanistic devices and on their susceptibility to group specific inhibitors. The cysteine proteinases, which were previously known as thiol proteinases, constitute the group of endopeptidases whose members rely for catalytic activity on the presence of a thiol group of a cysteine residue in the enzyme molecule. The other three main classes of endopeptidase are serine proteinases, aspartic proteinases, and metalloproteinases. Although not all cysteine proteinases have been subjected to substantial mechanistic study, those that have, appear to contain a thiol–imidazole interactive system within the catalytic site and it seems probable that this could be a common feature of all enzymes in the group. A plausible component of this interactive system is a thiolate–imidazolium ion-pair, the thiolate anion of which becomes transiently acylated during catalysis, assisted by general acid catalysis provided by the imidazolium ion.

Isolation and characterization of the four major cysteine-proteinase components of the latex of carica papaya L. reactivity characteristics towards 2,2′-dipyridyl disulfide of the thiol groups of papain, chymopapains A and B, and papaya peptidase A

High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A.

The kinetic analysis of hydrolytic enzyme catalyses: Consequences of non-productive binding.

for which the evidence in favour of this type d kinetic mechanism is particularly convincing is papain (EC 3.4.4.10). Thus Lowe and Williams [8] convincingly demonstrated that the papain-catalysed hydrolysis of methyl thionohippurate invoives the formation of an acyl-enzyme intermediate in which the acyl moiety of the substrate is linked to the active centre cysteine residue by a thiol ester bone. One of the major problems in the study of enzyme catalyses described by eq. (1) is the isolation of values for the first order rate constants for both acylation (k2) and deacylation (k3) or at least the assessment of their relative magnitudes. The recent approaches to the solution of this problem by Whitaker and Bender [9] and by Sluyterman [lo] yield conflicting results when applied to papain-catalysed hydrolyses. Whitaker and Bender determined k2 and k3 separately for the papain-catalysed hydrolysis of BAEE * and

The case for assigning a value of approximately 4 to pKaI of the essential histidine-cysteine interactive systems of papain, bromelain and ficin

One of the pKa values that characterizes the pHdependence of the kinetic parameters of reactions catalysed by the thiol proteases papain (EC 3.4.22.2.) bromelain (EC 3.4.22.4.) and Iicin (EC 3.4.22.3.) is near to 4 [l-S]. All three enzymes each possess a histidine imidazole group within 5A of their essential thiol group [6]. Of these enzymes, only papain is well characterized structurally: its essential (and only) thiol group (cysteine-25) is 7.5A from the carboxyl side chain of aspartic acid-158 and 3.48, from the N-l of the imidazole group of histidine-159 [7]. It is commonly assumed that a particular state of ionization of one or other of these two groups is crucial to the catalytic process and that it is the ionization of this group (pKa 4) that is reflected in the pH-dependence of the kinetic parameters. There is continuing discussion of whether this essential group of pKa 4 should be assigned to aspartic acid158 or to histidine-159. A recent paper by Murachi and Okumura [S] claims to show that the imidazolium ion of histidine-159 of papain and the essential imidazolium ion of bromelain are characterized by ‘normal’ pKa values near to 7 and thus are unlikely candidates for the essential groups of pKa 4 in these

On the mechanism of the α‐chymotrypsin‐catalysed hydrolysis of 4‐cis‐benzylidene‐2‐phenyloxazolin‐5‐one: Evidence for covalent non‐productive binding

During recent years the use of 4-c&benzylidene-2phenyloxazolin+one * (CBPO) and its derived methyl ester, methyl &enzamido-cis-cinnamate, as substrates for o-chymotrypsin has been the subject of a number of papers both from this laboratory [l-4] and from that of Zerner [5,6]. The purpose of this communication is to point out apparent inconsistencies in the results reported from the two laboratories, to confirm both sets of results, and to propose an interpretation which both rationalises the two sets of data and suggests interesting mechanistic consequences of the apparent inconsistencies. To initiate studies on the cw-chymotryptic hydrol-, ysis of substrates which possess bothanN-acylamino side chain and the chromophoric cinnamoyl moiety, we measured the rate of hydrolysis catalysed by & chymotrypsin of methyl &enzamido-cis-cinnamate. The value of kcat at 25.0” in phosphate buffer, I = 0.1, pH 7.9 containing 4.8% v/v dioxan was found to be

Evidence from two-protonic-state reactivity probe kinetics that chymopapain in fresh nonfruit latex ofCarica papaya consists of multiple forms of chymopapain A. The value of catalytic site characteristics in the identification, classification, and characterization of the papaya cysteine proteinases papain, the chymopapains, and papaya proteinase Ω

Fresh latex ofCarica papaya was collected from the stem, leaves, and petioles of the growing plant and fractionated by ion-exchange chromatography on a column of SP-Sephadex-C50 and by FPLC using a Mono S column. The fractions were examined for catalytic activity using Z-Lys-ONp andl-BAPNA as substrates and the thiol contents and reactivity characteristics were determined by using 2,2′-dipyridyl disulfide as a two-protonic-state thiol titrant and reactivity probe. By these methods the fresh nonfruit latex was shown to contain papain (EC 3.4.22.2), multiple forms of chymopapain, all of which have catalytic site reactivities characteristic of chymopapain A, and papaya proteinase Ω (originally called papaya peptidase A). The necessity now to characterize the catalytic site of a chymopapain in order to identify it is discussed.