Role of Lys159 in the Proton Relay System of the 3α‐hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

The role of Lys159 in the proton relay system of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase is studied through mutagenesis, chemical rescue, and solvent kinetic isotope effect to probe the proton transfer in catalysis. Mutants of K159A decrease the catalytic activity at 5 mM Caps, pH 10.4 by 20 and 120 fold in k cat and k cat /K m , respectively. The decreased rate can be rescued by the exogenous proton acceptor, such as buffers, small primary amines, and azide. The Brønsted relationship between the log( k cat / K d‐base ) of the external amine (corrected for molecular size effects) and p K a is linear for the K159A mutant‐catalyzed reaction at pH10.4 (β = 0.85 ± 0.09). The solvent kinetic isotope effect obtained for the wild‐type enzyme at p(L) 10.4 is 2.1 on k cat , indicating that an intramolecular proton transfer is involved in the reaction. No solvent kinetic isotope effect on k cat of the K159A mutant enzyme is observed at saturating Caps but a value of 4 on k cat is observed at 5 mM Caps at p(L) 10.4. A linear proton inventory on k cat is observed for K159A mutant with a transition state fractionation factor of around 0.24. These results indicate a single proton transfer to the external base with a late transition state occurred in a rate‐limiting step, and further demonstrate the dual role of Lys159 in the 3α‐hydroxysteroid dehydrogenase/carbonyl reductase catalyzed reaction, i.e ., to lower the pK a of the general base Tyr155 and to serve as the proton shuttle in the enzyme catalysis.