Journal
BIORESOURCE TECHNOLOGY
Volume 98, Issue 10, Pages 1931-1939Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.biortech.2006.07.037
Keywords
cysteine proteinases; proton inventory; mechanism of action; HAR/PT diagram
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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, 58 which seems more likely to be developed across a pK(a)-value close to 4.00, connecting D-COO-(158) and/or N-O delta 1(157) with the positively charged H-N delta 1-H delta 1(159) and/or H-N delta 1-H delta 1(158) (papain/chymopapain or bromelain numbering), constitutes another novelty of this work. (c) 2006 Published by Elsevier Ltd.
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