Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Molecular basis of pH stability.
The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques. Disassembly of the cyclic pentameric structure of human 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+ ion concentration, and with an activation energy of 155 kJ mol-1. A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below. These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous 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, exhibits 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 discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.
Biological Laboratory, University of Kent, Canterbury, United Kingdom.
L W
LW
Ruddock
S P
SP
Ruston
S M
SM
Kelly
N C
NC
Price
R B
RB
Freedman
T R
TR
Hirst
D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008961:Models, Structural; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli