Evidence that the carboxy groups of Asp158 in papain and caricain have abnormally low pKa values and thus do not contribute the key ionisations with pKa 4 that generate catalytic competence.

Studies on a number of cysteine proteinases of the papain (EC 3.4.22.2) family using pyridyland pyrimidyldisulphide reactivity probes have demonstrated that the catalytic site (Cys)-S-/(His)-Im+H ion-pair states of these enzymes are produced at low pH where the enzymes are inactive and that catalytic competence is generated by protonic dissociation across pKa 4 [ 1-41. The assignment of the pKa 4 to the most obvious candidate, Asp158 (papain numbering), is not supported by studies on Aspl58Asn mutants of papain [5] and papaya proteinase R (caricain, EC 3.4.22.30) [6]. In the present work studies on the reactions of papain and caricain with 4-chloro-7nitrobenzofurazan [Nbf-CI] using pH-dependent kinetics, spectral analysis of the resulting enzyme derivatives and computer modelling suggest pK, values for the carboxy groups in papain and caricain to be 2.8 and < 2 (possibly ca. 1 S) respectively. Earlier studies on papain using Nbf-CI as a thiol-specific reactivity probe demonstrated abnormally high reactivity at low pH [7-91. In the present work, improved stopped-flow methodology, together with the availability of SKETCHER [lo] for incisive evaluation of multiprotonic state pH-k data and the pKa values for (Cys)-S-/(His)Im+H ion-pair formation now known to be 3.4 for papain [I] and 2.9 for caricain have allowed the characteristics of the pH-k profiles for the reactions of papain and caricain with Nbf-CI to be properly defined. The second-order rate constants (k) for the reactions of both enzymes in the pH range ca. 2-5 at 25°C in aqueous buffer (I 0.1, containing 6.7% v/v ethanol) were determined by stopped-flow kinetic analysis at 412nm in the case of papain (the isosbestic point of the pH-dependent spectra of the product S-Nbf-papain) and at 41 7nm in the case of caricain (the pH-independent value of h,, of SNbf-caricain). The reactions, which were shown to obey overall second-order kinetics at pH 3 and pH 4.5, were studied using [enzyme],=IOpM and [Nbf-CIl0=800~M. Initial rates (v) were determined from the linear increase in A,,, or A,,, recorded over times ranging from 10s to 4 min and values of k in M-' s.' were calculated by using the values of [enzyme],, mbf-CI E~ 2-1 1360 M-' cm" for the papain reactions and ~~ ' ,=8400 Mcm for the caricain reactions. Inspection of the molecular environments of Asp158 in the crystal structures of the two enzymes and modelling of the S-Nbf-enzyme products were carried out using the computational techniques described in [I I ] using principally CHARMM and QUANTA. The pH-k profile for the reaction of papain with Nbf-CI in acidic media cons-ists of a skeyed bell-shaped_curve (pKl=2.8, pKll=3.4, pKII=4.0; k, =97 M-' s-I, k, =2.5 M-' s-I, k,=l.9 M-' s-I). The major bell-shaped component bounded by pKl and pKIl is interpreted as reaction of the thiolate anion in the (Cys25)-S-/(Hisl59)-Im+H ionpair produced across pKll=3.4 with Nbf-CI in a minor ionization state (i.e. from crossed-over ionisations) assisted by the undissociated carboxy group of Asp158 and inhibited by the carboxylate anion of this residue. The accumulation of electron density on the nitro group in the transition-state for the formation of the Meisenheimer-type intermediate would be stabilised by hydrogen-bond donation by \ (Aspl 58)-C02H and destabilised by (Aspl 58)-CO2-. Computer modelling of the product, S-Nbf-papain, supports the possibility of these interactions. If the activation of Nbf-CI in its reaction with papain is by direct interaction, the only hydrogen bond donor able to donate a proton to the nitro group of Nbf-CI is (Asp158)-C02H and its pKa is thus determined as 2.8. In marked contrast to the reaction of Nbf-CI with papain, the reaction with caricain is not characterised by a bell-shaped pH-k profile. Rather, k increases with increase in pH along a succession of sigmoidal components (pK, <2 and probably ca. 1.5, pKll=2.9, the pKa of (C_ys)-S-/(His)-Im_'H ion-pair fo-rmation, pKlII_=3.9 and pKlV=5.8; k,=0.1 M-' < I , k,=0.08 M-'s-', k,=0.9 M-ls-', k4=13 M-' s-I). The difference between the forms of the pH-k profiles for the reactions of papain and caricain is suggested to be due to the different microenvironments of Asp1 58. Thus in papain one oxygen atom of the carboxy side-chain is exposed and available for interaction with the Nbf-CI ligand, the other being hydrogen bonded to its own backbone -NH and that of Ala136. In caricain, however, both oxygen atoms of the Aspl 58 side-chain accept two hydrogen bonds from both the backbone -NH and the side-chain -NH: of Lys137 in one case and from both the backbone NH and the side-chain -OH of Ser136 in the other. The additional hydrogen bonding of the carboxylate in caricain could account for the lower pKa value (ca. 1.5) than that of the analogous group in papain (pKa 2.8). Support for the hypothesis that both ionisation states of the sidechain of Asp158 in papain interact with Nbf-CI whereas neither ionisation state of the analogous side-chain in caricain has a major effect is provided by the electronic absorption spectra of the respective S-Nbf-derivatives. Thus h,,=4 17nm for the caricain derivative and does not vary with pH in the range 2.1-4.9. For the papain derivative, however, hm,=424nm when the Nbflabel is in the presence of (Asp158)-C02H and hm,=408nm when in the presence of (Aspl 58)-C02-.