Substituent groups additional binding sites

While the equations look the same at a first glance, some striking differences can be seen on a closer inspection. First, the vertebrate, but not the bacterial DHFR equations contain an electronic parameter in addition to lipophilicity and molar refractivity terms. Second, in the case of L. casei (eq. 137) the 5-position of the benzyl group does not at all contribute to biological activities. An explanation could be derived by a comparison of the 3D structure of L. casei DHFR with the E. coli DHFR structure. The active sites of both enzymes are more or less identical in the geometries of the protein backbone and the amino acid side chains. However, there is one significant difference E. coli DHFR contains a methionine side chain in the area where the 5-substituents bind, while there is a relatively rigid leucine side chain in the L. casei DHFR which obviously interferes with the 5-substituents. Therefore, the active site of L. casei DHFR is sterically more constrained and the positive lipophilicity and polarizability contributions of the 5-substituents are counterbalanced by their steric hindrance [432, 682]. 

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