Effective Charge on Acetylcholinesterase Active Sites Determined from the Ionic Strength Dependence of Association Rate Constants with Cationic Ligands

Hans Jürgen Nolte, Eberhard Neumann, Terrone L. Rosenberry

Research output: Contribution to journalArticle

153 Scopus citations

Abstract

The reaction of the specific fluorescent cationic ligand N-methylacridinium with the active site of 11S acetylcholinesterase from electric eel was monitored by temperature-jump relaxation kinetics at a variety of ionic strengths. The ionic strength dependence of the bimolecular association rate constant is analyzed with a Brønsted-Debye-Hückel expression and leads to estimates of the association rate constant at zero ionic strength of k12° = 1.1 × 1010M−1s−1at 25 °C and the net charge number of the enzyme active site of ZE = -6.3. The ionic strength dependence of the second-order hydrolysis rate constant kcat/ kapp for acetylthiocholine under steady-state conditions is also very pronounced and indicates a value of ZE = -9. Thus, a large effective negative charge on the enzyme active site appears to be a general characteristic of its interaction with cationic ligands. The ionic strength dependence of kcat/ kapp is identical with that of sodium chloride, sodium phosphate, and sodium citrate, thus ruling out any possibility that the phenomena arise from a specific, partially competitive binding of Na+ to the enzyme active site. Substitution of the calculated electrostatic parameters into theoretical equations indicates that the most significant effect of these ZE values is a 2-3 order of magnitude reduction in the rate constant for dissociation of the initial ligand-enzyme encounter complex; this decrease renders the bimolecular reaction diffusion controlled. The high value of k120 and the space requirements of six to nine charged groups suggest that regions of the enzyme surface area larger than the catalytic sites themselves are effective in trapping cationic ligands.

Original languageEnglish (US)
Pages (from-to)3705-3711
Number of pages7
JournalBiochemistry
Volume19
Issue number16
DOIs
StatePublished - Aug 1 1980

ASJC Scopus subject areas

  • Biochemistry

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