The quenching of protein fluorescence that occurs when dihydrofolate reductase is titrated with coenzymes (NADPH or NADP+, reduced or oxidized nicotinamide-adenine dinucleotide phosphate, respectively) and substrates (folate or dihydrofolate) has been utilized to determine the stoichiometry and dissociation constants of the various complexes. The enzyme binds these reactants in a molar ratio of 1:1 characterized by dissociation constants ranging from 5X10-8 m to 9X10-7 m. An enzyme-NADPH complex can also be demonstrated from the effect of the enzyme on the fluorescence spectrum of NADPH; the emission maximum is shifted from 465 to 445 mμ with concomitant enhancement of fluorescence intensity. The folate analogs methotrexate and triampterene also quench the fluorescence of the enzyme. The binary complex of enzyme and triampterene is characterized by a dissociation constant of 3.4X10-7 m while the dissociation of methotrexate was characterized by a constant with a value less than 10-8 m. A ternary complex between NADPH, enzyme, and either inhibitor was observed. When NADPH and triampterene form a ternary complex, mutual stabilization of binding occurs that results in a 25-60-fold increase in the affinity of the enzyme for both the coenzyme and inhibitor over the affinity measured in binary complexes.
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