Heat in Activation Studies of 15-Lipoxygenase Isoenzyme-1
Abstract
Since the quaternary structure of soybean 15-lipoxygenase isoenzyme-l
has been elucidated by X-ray crystallography, attempts were made to
characterize of the enzyme further. Scanning calorimetry studies have
revealed that there is a thermal destabilization of the enzyme at 650C.
One of our long term goals is to determine which amino acid residues or
secondary structures contribute to the stabilization at the transition
temperature via site directed mutagenesis. To establish a basis for this long
term goal, the present study investigates the effects are observed as the
enzyme is exposed to its melting temperature of 65 0C and 70 0C for varying
time intervals. It was found that at 65 0C the enzyme activity reached 50%
inhibition after heating at approximately 20 minutes. Heating the enzyme at
70 0C for approximately 3 minutes resulted in 50% inhibition. Low
temperature Electron Paramagnetic Resonance (EPR) was also employed to
determine whether the ferric center was stabilizing the enzyme at the
transition temperature. We noted that there was an overall decrease in the
peak intensity with increasing duration of heat application which implies that
the enzyme was not activated to its catalytically active Fe3+ form upon
addition of 1 equivalent of product. Changes in the line shapes were
investigated in order to determine if the strain, caused by the heat
application, produced a distortion around the octahedral ferric center. This
would imply that the ferric center was stabilizing the enzyme. These changes
were not evident, however. We conclude that heating the enzyme for various
durations at 65 0C results in destabilizing of the enzyme in terms of
disruption of the weak intermolecular forces, for short heat applications, and
disruption of strong intermolecular forces to a point of irreversible unfolding
for longer heat applications. The EPR data support the destabilization but
evidence of stabilization by the iron is not conclusive.