It was also observed that 1,2-dehydrogenation can proceed even if a 2 position is substituted by a hydroxy group . This suggested that 3-ketosteroid can also be activated by an abstraction of the equatorial 2H atom by Y363. We decided to test if such activation yields a kinetically accessible energy profile. To abstract the 2H atom, Y363 has to break the H-bond with Y118, which in turn forms an H-bond with the 3-keto group (Figure ). Despite the increased nucleophilic character of the tyrosyl ion, the TS1 is reached at the later stage and the barrier is higher by 3 kcal/mol compared to the barrier of 2βH and also higher than the barrier of hydride transfer (Figure ). This demonstrates that although the enzyme has a preference for the abstraction of the axial 2βH, the enantioselectivity is not obligatory. * Activity assay required the addition of cyclodextrin, which makes it incomparable to the assay without one .