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Insight into the catalysis of hydrolysis of four newly synthesized substrates by papain: a proton inventory study.

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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Theodorou LG,Lymperopoulos K,Bieth JG,Papamichael EM

doi

10.1021/bi001615b

subject

Has Abstract

pub_date

2001-04-03 00:00:00

pages

3996-4004

issue

13

eissn

0006-2960

issn

1520-4995

pii

bi001615b

journal_volume

40

pub_type

杂志文章

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