The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.

We describe the synthesis of several polyamides that retain the secondary structure of proteins and contain derivatizable side chains. The derivatizable side chain allows for further reaction of the polymer chain (e.g., chain cross-linking or addition of pendant groups). Polymers of alpha-amino acids containing a terminal unsaturated bond on the side chain have been synthesized. Poly-L-pentenyl glycine, poly-L-propargyl glycine, and poly-L-allyl glycine were synthesized chemically via Leuchs' anhydrides and enzymatically using subtilisin Carlsberg. Poly-L-propargyl glycine and poly-D,L-allyl glycine folded into the beta-sheet configuration whereas poly-L-pentenyl glycine assumed a helical conformation. The secondary structure of poly-L-allyl glycine and poly-D,L-pentenylglycine could not be determined conclusively. Comparison of properties between the polymers obtained chemically and enzymatically is provided.

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