The catalytic mode of cysteine proteinases of papain (C1) family

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–(kcat/Km) profiles, and it was found: (a) kcat/Km = k1, and thus KS = k2/k1 is a dynamic equilibrium constant, (b) bowed-downward PI for kcat/Km exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for KS, k2 and k3. 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 pKa-value close to 4.00, connecting DCOO-158 and/or NOδ1157, with the positively charged HNδ1-Hδ1159 and/or HNδ1-Hδ1158 (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.