help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

Biophysical Journal 65: 215-226 (1993)
© 1993 the Biophysical Society

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, S J
Right arrow Articles by Barkley, M D
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, S J
Right arrow Articles by Barkley, M D

Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase.

S J Kim, F N Chowdhury, W Stryjewski, E S Younathan, P S Russo and M D Barkley

Department of Chemistry, Louisiana State University, Baton Rouge 70803.

ABSTRACT

The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.




This article has been cited by other articles:


Home page
 Protein Sci.Home page
D. S. Libich, C. M.D. Hill, I. R. Bates, F. R. Hallett, S. Armstrong, A. Siemiarczuk, and G. Harauz
Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+-calmodulin: Effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism
Protein Sci., July 1, 2003; 12(7): 1507 - 1521.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
E. Feinstein, G. Deikus, E. Rusinova, E. L. Rachofsky, J. B. A. Ross, and W. R. Laws
Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics
Biophys. J., January 1, 2003; 84(1): 599 - 611.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. M. Byrnes, W. Hu, E. S. Younathan, and S. H. Chang
A Chimeric Bacterial Phosphofructokinase Exhibits Cooperativity in the Absence of Heterotropic Regulation
J. Biol. Chem., February 24, 1995; 270(8): 3828 - 3835.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. L. Kimmel and G. D. Reinhart
Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus
PNAS, April 11, 2000; 97(8): 3844 - 3849.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1993 by the Biophysical Society.