The cAMP-dependent protein kinase (PKA) mediates a myriad of cellular signaling events and its activity is tightly regulated both in space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted either by interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of myristoylated PKA-C steers the enzyme towards lipids independent of its regulatory subunit or an A-kinase anchoring protein (AKAP), providing an additional mechanism to localize the enzyme near membrane-bound substrates.

The asymmetric reduction of benzylic and heteroaryl ketones to the corresponding (R)‐alcohols using I86A Thermoanaerobacter ethanolicus alcohol dehydrogenase (I86A TeSADH) is described. This single amino acid mutation not only makes the active site of I86A TeSADH able to accommodate more sterically demanding substituents than those accommodated by wild‐type TeSADH, but it also reverses the substrate stereospecificity of TeSADH.

The ability to control the substrate specificity and stereochemistry of enzymatic reactions is of increasing interest in biocatalysis. As this review highlights, this control can be achieved through various means, including mutagenesis of active site residues, alteration of physical variables like temperature and pressure, as well as through changing the reaction medium. While the focus of this article is on alcohol dehydrogenase reactions, each of these techniques can be readily applied towards other enzyme classes as well.In order to understand better the orientation of the substrate in the enzyme active site, it is important to have a map of the active site. In pioneering studies of the 1960's, Prelog proposed using a diamond lattice structure to visualize oxidoreductase active sites. 2 To validate his hypothesis, he tested the reaction with a series of increasingly larger substrates, and was able to determine the maximum spatial limits of the active site in each direction. By overlaying the molecules, which gave successful results, the active site was effectively mapped. It was important to have a means to map the active site, since this would give a measure of the limit of stereospecificity, and in the future, substrate specificity.In an extension of Prelog's diamond lattice model, Jones and coworkers further studied its utility towards horse liver alcohol dehydrogenase (HLADH) reactions. 3-7 In the early 1980's, Jones and Jakovac proposed a cubic space section model, due to the disadvantages of the diamond lattice for mapping an active site. 3 Among the disadvantages were the use of sp 3 hybridized carbon bond lengths and angles which could invariably limit researchers to use the diamond lattice model to choose substrates for analysis. With the cubic space section model, the cube sizes were flexible, which allowed researchers to use blocks that were as large or small as desired. The results for simpler examples, which worked adequately with Prelog's diamond lattice, also worked satisfactorily with the cubic model. Jones and coworkers highlighted further examples that fit nicely with the new cubic shaped model. [4][5][6][7] While mutagenesis can be accomplished routinely at present, it was not always the case. Hence, Jones studied the varying active sites of naturally occurring variants. Even though the cubic space model was useful, it was not widely adopted by other research groups due to the advent of more straightforward X-ray crystal structures, which give very clear information regarding the shape of the active site. Nonetheless, these early active site models have paved the way for

An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than 2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris buffer/acetone (90:10, v/v).