Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and Michaelis-Menten concepts.
Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] >> k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.
Lee
Hye Jin
HJ
Department of Chemistry, University of California-Irvine, Irvine, CA 92697, USA.
Wark
Alastair W
AW
Goodrich
Terry T
TT
Fang
Shiping
S
Corn
Robert M
RM
D000327:Adsorption; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000032:analysis; D004798:Enzymes / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature
D004798:Enzymes; D004800:Enzymes, Immobilized; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III