Web Release Date: September 10,
Determination of Ribonuclease H Surface Enzyme Kinetics by Surface Plasmon Resonance Imaging and Surface Plasmon Fluorescence Spectroscopy
Department of Chemistry, University of California-Irvine, Irvine, California 92697
Received for review July 20, 2005. Accepted August 22, 2005.
Abstract:
The kinetics of the ribonuclease H (RNase H) surface
hydrolysis of RNA-DNA heteroduplexes formed on DNA
microarrays was studied using a combination of real-time
surface plasmon resonance imaging (SPRI) and surface
plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model
that couples diffusion, enzyme adsorption, and surface
enzyme kinetics. This model is characterized by a set of
three rate constants, enzyme adsorption (ka), enzyme
desorption (kd), enzyme catalysis (kcat), and one dimensionless diffusion parameter (
). Values of ka = 3.15
(±0.20) × 106 M-1·s-1, kd = 0.10 (±0.05) s-1, and kcat
= 0.95 (±0.10) s-1 were determined from fitting all of
the SPRI and SPFS data sets. One of the most interesting
kinetic parameters is the surface RNase H hydrolysis
reaction rate constant (kcat), which was found to be ~10
times slower than that observed in solution, but ~100
times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA
microarrays (kcat = 0.009 s-1). Moreover, the surface
coverage of the intermediate enzyme-substrate complex
(ES) was found to be extremely small during the course
of the reaction because kcat is much larger than the
product of ka and the bulk enzyme concentration.
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