Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase

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Abstract
Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methrotrexate concentration indicates the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 .times. 107 M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 .times. 105 M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1. These two isomerizations transform a modest association constant for initial binding into an extraordinary high one (1.9 .times. 1010 M-1) and are therefore the key mechanism for tight binding. The rate constant for reversal of the slow isomerization is 0.0062 min-1, corresponding to t1/2 = 1.9 h, so that release of MTX from its complex with SFDHFR II can occur no faster than this at pH 7.3 and 20.degree. C. The slow isomerization does not seem to occur in binary complexes. Binary complex formation by MTX and dihydrofolate reductase from Lactobacillus casei is biphasic, but this is due to the presence of two forms of the enzyme which bind MTX at different rates. This may obscure possible isomerization of binary complexes. Isoenzyme I of SFDHFR shows a slower phase due to isomerization of the binary complex of pH 6.0 but not a higher pH.

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