Insight into the Catalysis of Hydrolysis of Four Newly Synthesized Substrates by Papain: A Proton Inventory Study

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• 作者：Leonidas G. Theodorou ; Kostas Lymperopoulos ; Joseph G. Bieth ; and Emmanuel M. Papamichael
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 3, 2001
• 年：2001
• 卷：40
• 期：13
• 页码：3996 - 4004
• 全文大小：123K
• 年卷期：v.40,no.13(April 3, 2001)
• ISSN：1520-4995

文摘

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 inventorymethod to their hydrolysis by papain. Useful relationships between the rate constants of the catalyticreaction 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 k₁, k_-1, and k₂ were estimated. Moreover,it was found that k_cat/K_m = k₁ for all four substrates, while two exchangeable hydrogenic sites, one in theground state and another in the transition state, generate an inverse isotope effect during the reactiongoverned by this parameter. The proton inventories of both k₂ and k₃ are essentially linear, whatever theacyl moiety and/or the leaving group of the substrate. The proton inventories of K_S are also essentiallylinear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from asingle hydrogenic site in the product state. This latter, in agreement to both the small transition statefractionation factors found for k_cat/K_m (or k₁) and the unit ground-state fractionation factors found for k₂,argues for the formation of a tetrahedral adduct during the reaction governed by the k₁ parameter.Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceedthrough similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movementof one proton.