Stuart R. Stone

Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogues

Different classes of folate analogues have been examined with respect to the mechanism of their inhibition of dihydrofolate reductases from Escherichia coli and chicken liver. In addition, the degree of synergism between the binding of these compounds and NADPH has been investigated. Methotrexate acts as a slow, tight-binding inhibitor of both enzymes whereas trimethoprim is a slow, tight-binding inhibitor of the enzyme from E. coli and a classical inhibitor of the chicken-liver enzyme. Pyrimethamine, 2,4-diamino-6,7-dimethylpteridine, a phenyltriazine, folate and folinate exhibit classical inhibition. The degree of synergism between the binding of NADPH and the inhibitor varied from low for pyrimethamine and folate to very large for the phenyltriazine which binds to the chicken-liver enzyme almost 50 000-times more tightly in the presence of NADPH. The degree of synergism is reflected in the type of inhibition that the folate analogues yield with respect to NADPH. Compounds which exhibit slight synergism give noncompetitive inhibition whereas those with a high degree of synergism yield uncompetitive inhibition. With the exception of folinate, all compounds that act as classical inhibitors give rise to competitive inhibition with respect to dihydrofolate. Folinate exhibits competitive inhibition against NADPH and noncompetitive inhibition against dihydrofolate. These results are consistent with the formation of an enzyme-dihydrofolate-folinate complex. The (6S, alphaS)-diastereoisomer of folinate was bound at least 1000-times more tightly than the (6R, alphaS)-diastereoisomer. Consideration has been given to the possible interactions that occur between residues on the enzyme and groups on the inhibitor that give rise to slow-binding inhibition.

Inhibition of dihydrofolate reductase from bacterial and vertebrate sources by folate, aminopterin, methotrexate and their 5-deaza analogues

The inhibition of dihydrofolate reductases from Escherichia coli and chicken liver by folate, methotrexate, aminopterin and their 5-deaza analogues was investigated to examine the importance of the N-5 nitrogen in slow-binding inhibition. Methotrexate, aminopterin and their 5-deaza analogues acted as slow, tight-binding inhibitors of both enzymes. Inhibition by methotrexate and 5-deazamethotrexate conformed to a mechanism in which there is an initial rapid formation of an enzyme-NADPH-inhibitor complex followed by a slow isomerization of this complex (Mechanism B). Aminopterin exhibited the same type of inhibition with the enzyme from E. coli. With the chicken-liver enzyme, however, the inhibition by aminopterin conformed to another type of slow-binding mechanism which involves only the slow interaction of the inhibitor with the enzyme to form an enzyme-NADPH-inhibitor complex (Mechanism A). The inhibition of both enzymes by 5-deazaaminopterin was also described by Mechanism A. Folate behaved as a classical, steady-state inhibitor of both enzymes, whereas 5-deazafolate exhibited slow-binding inhibition (Mechanism B) with the enzyme from E. coli and classical, steady-state inhibition with the enzyme from chicken liver. The substitution of a carbon for a nitrogen at the 5-position of methotrexate and aminopterin did not affect the tightness of binding of these compounds. By contrast, 5-deazafolate was bound about 4000 times more tightly than folate to the enzyme from E. coli and about 30 times more tightly than folate to the chicken-liver enzyme. Reasons for the differences in the binding of folate and 5-deazafolate are discussed.

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.

The interaction of an ionizing ligand with enzymes having a single ionizing group: Implications for the reaction of folate analogues with dihydrofolate reductase

Binding theory has been developed for the reaction of an ionizing enzyme with an ionizing ligand. Consideration has been given to the most general scheme in which all possible reactions and interconversions occur as well as to schemes in which certain interactions do not take place. Equations have been derived in terms of the variation of the apparent dissociation constant (Kiapp) as a function of pH. These equations indicate that plots of pKiapp against pH can be wave-, half-bell- or bell-shaped according to the reactions involved. A wave is obtained whenever there is formation of the enzyme-ligand complexes, ionized enzyme . ionized ligand and protonated enzyme . protonated ligand. The additional formation of singly protonated enzyme-ligand complexes does not affect the wave form of the plot, but can influence the shape of the overall curve. The formation of either ionized enzyme . ionized ligand or protonated enzyme . protonated ligand, with or without singly protonated enzyme-ligand species, gives rise to a half-bell-shaped plot. If only singly protonated enzyme-ligand complexes are formed the plots are bell-shaped, but it is not possible to deduce the ionic forms of the reactants that participate in complex formation. Depending on the reaction pathways, true values for the ionization and dissociation constants may or may not be determined.

The effect of anions on the reaction catalyzed by lupine-nodule glutamate dehydrogenase

Abstract The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase ( l -glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH 4 ) 2 SO 4 but when NH 4 Cl or NH 4 -acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH 4 Cl and NH 4 -acetate, however, if the concentration of Cl − or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH 4 + and the apparent substrate inhibition by NH 4 Cl and NH 4 -acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I − caused parabolic noncompetitive inhibition with respect to NH 4 + and NO 3 − caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl − was a linear competitive inhibitor with respect to both NAD and l -glutamate whereas NO 3 − caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.