Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations

Abstract

The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically.

Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion

“…We have demonstrated, using a variety of experimental approaches, the value of using natural variants of the papain family of cysteine proteinases to reveal gradations in functional characteristics and mechanistic phenomena that are more easily demonstrated in some variants than in others [1][2][3][4]. A recent study [5] provided evidence of a particularly marked difference in observed features of catalytic mechanism between papain (EC 3.4.22.2) and caricain [papaya (Carica papaya) proteinase (EC 3.4.22.30)] on the one hand and actinidin (EC 3.4.22.14) and ficin (EC 3.4.22.3) on the other. Papain is the much studied minor cysteine proteinase component of the latex of C. papaya and caricain a major companion cysteine proteinase component of the same latex.…”

“…Evidence for the existence of ES and ES , tetrahedral species between ES and ES and between ES and E + P 2 , and electrostatic modulation of catalytic-site geometry required to promote the acylation reaction is discussed in [1][2][3][4] and summarized in [5]. Additional modulation by specific binding interactions is discussed, e.g., in [7].…”

Characterization of the acidic and basic limbs of a bell‐shaped pH profile in the inhibitory activity of bromelain inhibitor VI

“…In this study, we examined the pH dependence of the inhibitory activity against bromelain and another cysteine proteinase, papain, which differs markedly from bromelain in certain properties. 20 As shown in Figure 6, the optimal inhibitory pH against both proteinases was almost the same, ca. 4.0, but the overall inhibitory activity toward papain was shown to be a little weaker than that toward bromelain.…”

“…There is interesting evidence of a particularly marked difference in the observed features of the catalytic mechanisms between papain and caricain on the one hand and actinidin and ficin on the other. 20 For example, in the case of actinidin, the pH dependence of k cat /K m for the catalyzed hydrolysis of -N-benzoyl-L-arginine-p-nitrophenol depends on three pK a values: 3.1-3.2 and 5.0-5.2 on the enzyme acidic limb and 9.5 on the basic one 26 ; for papain, on the other hand, it depends on a set of the values 3.7-4.0 on the enzyme acidic limb and 8.1-8.3 and possibly approximately 9.5 on the basic one. 27 Bromelain belongs not to the papain subfamily but to the actinidin one with respect to the pH dependence of the second-order rate constants.…”

“…Furthermore, it was reported that the reaction mechanisms of bromelain and ficin resemble that of actinidin. 20,28 Here, we discuss which residue(s) in the actinidin subfamily are related to the hydronic state of an ionizable group(s) with a pK a of 5-6. As shown in Figure 8, the Asp55-Arg96 ion pair in the residue numbering of papain might be a possible pK II candidate for the actinidin subfamily.…”