Atypical kinetics of immobilized firefly luciferase

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Abstract
The kinetic properties of collagen-bound firefly luciferase have been investigated. Under definite hydrodynamic conditions with low agitation in the reaction medium, the observed behavior is modified compared to the enzyme free in solution: reducing the stirring rate decreases the observed enzymatic activity. But diffusional resistances alone cannot account for these atypical kinetics though mass transfer may certainly play an important role during the transient state of the bioluminescent reaction. After immobilization, the time necessary to reach the steady state increased from 300 ms to 3 min and the two substrates, luciferin and ATP, behave differently with respect to the enzyme: The nature of the saturating substrate first in contact with the bound enzyme is not indifferent suggesting that immobilization can reveal behaviors or mechanisms which are not visualized with the free enzyme.