Enzymatic dynamics of individual Beta-Galactosidases

PHYS 624

Brian P. English1, A. M. van Oijen1, Kang Taek Lee1, Guobin Luo1, Hongye Sun2, and X. Sunney Xie1. (1) Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, (2) Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404
A novel single-molecule enzymatic assay is developed that employs the conversion of a non-fluorescent enzymatic substrate into a brightly fluorescent product to observe dynamics in the turnover rate of an individual enzyme. The continuous replenishing of fluorescent signal considerably extends the observable timescale that previously was limited by photobleaching in single molecule assays. Here we present the direct observation of the catalytic activity of individual E.coli beta-galactosidase enzymes. The catalytic conversion of a non-fluorescent resorufin-beta-D-galactopyranoside substrate molecule into fluorescent resorufin is reflected by a short fluorescence burst before the fluorescent product diffuses out of the laser focus. The autocorrelation function of the fluorescence intensity exhibits highly stretched exponential behavior, revealing large fluctuations in catalytic rate on timescales longer than the enzymatic cycle. This is attributed to the presence of many long-lived protein conformers undergoing interconversion over a broad range of timescales.

Physical Chemistry Poster Session
7:30 PM-10:00 PM, Wednesday, August 25, 2004 Pennsylvania Convention Center -- Hall D, Poster

8:00 PM-10:00 PM, Monday, August 23, 2004 Pennsylvania Convention Center -- Hall D, Sci-Mix

Division of Physical Chemistry

The 228th ACS National Meeting, in Philadelphia, PA, August 22-26, 2004