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Chemical modification of the RTEM‐1 thiol β‐lactamase by thiol‐selective reagents: Evidence for activation of the primary nucleophile of the β‐lactamase active site by adjacent functional groups
Author(s) -
Knap Anna K.,
Pratt R. F.
Publication year - 1989
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340060314
Subject(s) - thiol , nucleophile , chemistry , cysteine , active site , stereochemistry , iodoacetamide , reagent , hydrogen bond , leaving group , medicinal chemistry , enzyme , organic chemistry , molecule , catalysis
The RTEM‐1 thiol β‐lactamase (Sigal, I. S., Harwood, B. G., Arentzen, R., Proc. Natl. Acad. Sci. USA. 79:7157–7160, 1982) is inactivated by thiol‐selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4′‐dipyridyldisulfide, which modify the active site thiol group. The pH‐rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH 2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The p K a of the active site thiol is therefore shown by the data to be below 4.0. This low p K a is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH 2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol β‐lactamase is stabilized by two hydrogen‐bond donors. One of these, of p K a greater than 9.0, is suggested to be the conserved and essential Lys‐73 ammonium group, of p K a around 6.7, is less clear, but may be the conserved Glu‐166 carboxylic acid. β‐Lactamase activity is associated with the EH 2 form, and thus the β‐lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol β‐lactamase) and two acidic (hydrogen‐bond donor) groups (one of which is likely to be the above‐mentioned lysine ammonium group).