Studies on the active site of pig kidney aldehyde reductase.

observed in enzyme-methotrexate complexes. The orientation that appears to be required for catalysis can be readily achieved by rotations of the pteridine of 180° around the C-6-C-9 bond and 30° about the C-9-N-10 bond. In this orientation, C-4 of the NADPH nicotinamide ring closely approaches C-6 of the substrate, and N-5 of the substrate is adjacent to aspartic acid-30 of the enzyme. It has been suggested that in this orientation either aspartic acid-30 or threonine136 may provide the proton that is transferred to N-5 (Matthews et al., 1978). Comparative studies on the structures and kinetic properties of two isozymes (forms 1 and 2) of dihydrofolate reductase from E. coli RT5OO (Baccanari et al., 1981) provide further information regarding the mechanism of action of these enzymes. Although the amino acid sequences of these two isoenzymes differ only at position-3 1, which is arginine in form 2 and leucine in form I, they do exhibit considerably different inhibitor-binding and kinetic properties. From equilibriumdialysis studies it has been shown that the inhibitors methotrexate and trimethoprim bind more tightly to form 1 than to form 2. The isoenzymes also differ in their catalytic properties, form 1 having a 10-fold higher catalytic-centre activity (turnover number) at pH 7 than form 2. In the presence of Ba2+, which is a powerful inhibitor of the form-1 isoenzyme but a much weaker inhibitor of the form-2 isoenzyme, the properties of form 1 resemble those of form 2. It has been suggested that the interaction of arginine-31 and other residues in the active-site region could account for the characteristic properties of the form-2 enzyme and that a similar interaction with Ba2+ can alter the properties of the form 1 enzyme to those resembling form 2.