Isosteric and non-isosteric modification of carboxyl groups of papain

C. Kooistra, L.A.A.E. Sluyterman

Research output: Contribution to journalArticleAcademic

3 Citations (Scopus)


Guanidinated mercuri-papain (Gu-papain) was reacted with N-ethylbenziosoxazolium tetrafluoroborate at pH 4.2, 0°C, to yield highly reactive N-ethylsalicylamide esters. On varying the amount of reagent applied 2.5–10 car¿yl groups were modified. Appropriate plotting of the data indicated that all 12 groups exposed in the X-ray structure were modified to an extent of 80% in the final preparation, concomitant with a similar loss of activity towards Na-benzoyl-l-arginine ethyl ester. The preparations regained complete activity on saponification of the ester groups and removal of some oligomeric material by gel filtration. Considerable activity was recovered when the ester groups were completely replaced by amide groups by subjecting the esters to ammonolysis in 2 M ammonium acetate/ammonia (pH 9.2). The final preparation, after gel filtration, exhibited Km = 57 ± 1 mM and kcat = 26 ± 0.2 s-1 towards BAEE (native papain Km = 18 mM and kcat = 26 s-1. It may be concluded that replacement of a bulky modifying group by an isosteric one may cause considerable recovery of activity, emphasizing the importance of isostericity in suppressing the ionizing ability of ionizable groups; furthermore, that a large shift in overall charge, caused by amidation of all accessible car¿yl groups, does not affect the catalytic steps. The absence of effect of side-chain charges on the ion pair in the active site is briefly discussed.
Original languageEnglish
Pages (from-to)115-120
Number of pages6
JournalBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology
Issue number1-2
Publication statusPublished - 1989

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