Abstract:
The spatial limits of the active site in the benzylic hydroxylase
enzyme of the fungus Mortierella isabellina were investigated. Several
molecular probes were used in incubation experiments to determine the
acceptability of each compound by this enzyme. The yields of benzylic
alcohols provided information on the acceptability of the particular
compound into the active site, and the enantiomeric excess values
provided information on the "fit" of acceptable substrates.
Measurements of the molecular models were made using Cambridge
Scientific Computing Inc. CSC Chem 3D Plus modeling program.
i
The dimensional limits of the aromatic binding pocket of the benzylic
hydroxylase were tested using suitably substituted ethyl benzenes. Both
the depth (para substituted substrates) and width (ortho and meta
substituted substrates) of this region were investigated, with results
demonstrating absolute spatial limits in both directions in the plane of the
aromatic ring of 7.3 Angstroms for the depth and 7.1 Angstroms for the
width. A minimum requirement for the height of this region has also
been established at 6.2 Angstroms.
The region containing the active oxygen species was also
investigated, using a series of alkylphenylmethanes and fused ring
systems in indan, 1,2,3,4-tetrahydronaphthalene and benzocycloheptene
substrates. A maximum distance of 6.9 Angstroms (including the 1.5
Angstroms from the phenyl substituent to the active center of the heme
prosthetic group of the enzyme) has been established extending directly in
ii
front of the aromatic binding pocket. The other dimensions in this region
of the benzylic hydroxylase active site will require further investigation to
establish maximum allowable values.
An explanation of the stereochemical distributions in the obtained
products has also been put forth that correlates well with the experimental
observations.
