Lw. Ruddock et al., KINETICS OF ACID-MEDIATED DISASSEMBLY OF THE B-SUBUNIT PENTAMER OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN - MOLECULAR-BASIS OF PH STABILITY, The Journal of biological chemistry, 270(50), 1995, pp. 29953-29958
The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (Et
xB) is highly stable, maintaining its quaternary structure in a range
of conditions that would normally be expected to cause protein denatur
ation, In this paper the structural stability of EtxB has been studied
as a function of pH by electrophoretic, immunochemical, and spectrosc
opic techniques, Disassembly of the cyclic pentameric structure of hum
an EtxB occurs only below pH 2. As determined by changes in intrinsic
fluorescence this process follows first-order kinetics, with the rate
constant for disassembly being proportional to the square of the H+ io
n concentration, and with an activation energy of 155 kJ mol(-1). A C-
terminal deletion mutant, hEtxB214, similarly shows first-order kineti
cs for disassembly but with a higher pH threshold, resulting in disass
embly being seen at pH 3.4 and below. These findings are consistent wi
th the rate-limiting step for disassembly of human EtxB being the simu
ltaneous disruption of two interfaces by protonation of two C-terminal
carboxylates. By comparison, disassembly of the B-subunit of cholera
toxin (CtxB), a protein which shows 80% sequence identity with EtxB, e
xhibits a much lower stability to acid conditions; with disassembly of
CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol(-
1). Reasons for the observed differences in acid stability are discuss
ed, and the implications of these findings to the development of oral
vaccines using EtxB and CtxB are considered.