Abstract: Two thermophilic extracellular proteases, designated Lmm-protease-Lh ( approximately 29 kDa) and Hmm-protease-Lh ( approximately 62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK(2). Both novel proteases maintained full activity at 60 degrees C after 1-h incubation at 10 degrees C as well as at 80 degrees C, showing optimum k(cat)/K(m) values at pH 7.00 and 60 degrees C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis-Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis-Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k(cat)/K(m)) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis-Menten parameters.

Abstract: The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.

Abstract: A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.

Abstract: We studied the role of D158 in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the kcat/Km parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for kcat/Km, as in papain, whereas for subtilisin we found "dome-shaped" PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D158 as part of a catalytic triad in papain-like cysteine proteinases.

Abstract: We synthesized the following four new peptide substrates, Suc-Phe-Leu-pNA, Suc-Phe-Leu-NMec, Suc-Phe-Leu-ONPh, and Pht-Phe-Leu-pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationships between the rate constants of the catalytic reaction have been established and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter K(S) and the rate constants k(1), k(-)(1), and k(2) were estimated. Moreover, it was found that k(cat)/K(m) = k(1) for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse isotope effect during the reaction governed by this parameter. The proton inventories of both k(2) and k(3) are essentially linear, whatever the acyl moiety and/or the leaving group of the substrate. The proton inventories of K(S) are also essentially linear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small transition state fractionation factors found for k(cat)/K(m) (or k(1)) and the unit ground-state fractionation factors found for k(2), argues for the formation of a tetrahedral adduct during the reaction governed by the k(1) parameter. Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceed through similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movement of one proton.