Dihydrofolate reductase hydride transfer

The NAD- and NADP-dependent dehydrogenases catalyze at least six different types of reactions simple hydride transfer, deamination of an amino acid to form an a-keto acid, oxidation of /3-hydroxy acids followed by decarboxylation of the /3-keto acid intermediate, oxidation of aldehydes, reduction of isolated double bonds, and the oxidation of carbon-nitrogen bonds (as with dihydrofolate reductase). 

Figure 10. The ternary complex of the enzyme dihydrofolate reductase, the substrate and the cofactor during the transition state of the hydride ion transfer. The enzyme backbone atoms are shown alone for clarity and are colored blue. The substrate is shown in yellow and the cofactor is in red. The bond colored in light blue indicates the hydride ion being shared by both the cofactor and the substrate before the transfer to the substrate. Water molecules around the residue pteridine of the substrate and the nicotinamide ring of the cofactor alone are shown and colored in light blue. The yellow spheres represent the sodium ions and the pink spheres the chloride ions.

Dihydrofolate reductase from E coir, hydride transfer from NADPH to 7,8-DHE... 

In a collaboration between the Abelson and Hecht labs [56b], a series of noncoded amino acids were introduced into dihydrofolate reductase (DHFR) to probe substrate binding and the requirement of an aspartic acid residue for catalytic competence. When aspartic acid analogs mono- or disubstituted at the )0-carbon were substituted for the active site aspartic acid residue, the mutant DHFRs were still able to catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate at 74 - 86 % of the wild-type rate. While hydride transfer from NADPH is not the rate-limiting step for the wild-type enzyme at physiological pH, a kinetic isotope experiment with NADPD indicated that hydride transfer had likely become the rate-limiting step for the mutant containing the )0,)0-dimethylaspartic acid. 

Two-dimensional heteronuclear ( H- N) nuclear magnetic relaxation studies indicate that the dihydrofolate reductase-folate complex exhibits a diverse range of backbone fluctuations on the time-scale of picoseconds to nanoseconds To assess whether these dynamical features influence Michaelis complex formation, Miller et al used mutagenesis and kinetic measurements to assess the role of a strictly conserved residue, namely Gly-121, which displays large-amplitude backbone motions on the nanosecond time scale. Deletion of Gly-121 dramatically reduces the hydride transfer rate by 550 times there is also a 20-times decrease in NADPH cofactor binding affinity and a 7-fold decrease for NADP+ relative to wild-type. Insertion mutations significantly decreased both... 

Fig. 14. Dihydrofolic acid. In the dihydrofolate reductase reaction, the double bond between N-5 and C-6 is reduced by hydride transfer from the 4-pro-R position of NADPH to C-6, and addition of a proton at N-5.

Hammes-Schiffer, S. Watney, J. B. Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase coupled motions and distal mntations, Philos. Trans. R. Soc. London Ser. B 2006, 361, 1365-1373. 

Garcia-Viloca, M., Truhlar, D. and Gao, J. (2003). Reaction-path energetics and kinetics of the hydride transfer reaction catalyzed by dihydrofolate reductase. Biochemistry 42, 13558-13575... 

Thorpe, I. and Brooks, C. (2004). The coupling of structural fluctuations to hydride transfer in dihydrofolate reductase. Proteins Struct. Fund. Bioinf. 57, 444-457... 

Rajagopalan, P. T. R., Lutz, S., Benkovic, S. J. (2002) Coupling interactions of distal Residues enhance dihydrofolate reductase catalysis mutational effects on hydride transfer rates. Biochemistry 41, 12618-12628. 

Maglia, G., Javed, M. H., Allemann, R. K. (2003) Hydride transfer during catalysis by dihydrofolate reductase from Thennotoga maritima, Biochem. J. 374, 529-535. 

Wong, K., Watney, J. B., Hammes-Schieeer, S. (2004) Analysis of electrostatics and correlated motions for hydride transfer in dihydrofolate reductase, J. Phys. Chem. B. 108, 12231-12241. 

Thorpe, I. E., Brooks, C. L. I. (2003) Barriers to hydride transfer in wild type and mutant dihydrofolate reductase fom E.coli, J. Phys. Chem. B. 107, 14042-14051. 

Wong, K., Selzee, T., Benkovig, S. J. (2004) Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase, Proc. Natl. Acad. Sci. USA, 102, 6807-6812. 

The use of pH variation and isotope effects in transient kinetics can be illustrated with a recent study on dihydrofolate reductase. Analysis by steady-state methods had indicated an apparent p/fa of 8.5 that was assigned to an active site aspartate residue required to stabilize the protonated state of the substrate (59). In addition, it was shown that there was an isotope effect on substitution of NADPD (the deuterated analog) for NADPH at high pH but not at low pH, below the apparent p/fa This somewhat puzzling finding was explained by transient-state kinetic analysis. Hydride transfer, the chemical reaction converting enzyme-bound NADPH and dihydrofolate to NAD+ and tetrahydrofolate, was shown to occur at a rate of approximately 1000 sec at low pH. The rate of reaction decreased with increasing pH with a of 6.5, a value more in line with expectations for an active site aspartate residue. As shown in Fig. 14, there was a threefold reduction in the rate of the chemical reaction with NADPD relative to NADPH. Thus direct measurement of the chemical reaction revealed the full isotope effect. 

Fig. 14. Isotope effect on the rate of hydride transfer. The rate of hydride transfer to dihydrofolate catalyzed by dihydrofolate reductase (IS /rAf) was measured by fluorescence energy transfer, exciting the protein at 280 nm and observing emission by NADPH at 450 nm. The reaetion with NADPH oecurred at a rate of 450 see", followed by a linear phase at 12 sec , as shown by the smooth line. The rate of the burst observed with NADPD, the deuterium analog, occurred at 150 sec . Reproduced with permission from (27).

Fio. 15. The pH dependence of a reaction catalyzed by dihydrofolate reductase. The observed rate of hydride transfer (- -) is compared with the rate of product release (—) and kcui—) on a log scale as a function of pH. The break in the rate of steady-state turnover at pH 8.5 is due to a change in the rate-limiting step from product release to hydride transfer. Reproduced with permission from (27). 

Surprisingly, however, recent evidence showed that dihydrofolate reductase is not a universal enzyme. About one-third of the sequenced eubacteria use a flavin-dependent thymidylate synthetase (FDTS) (ThyX protein, EC 2.1.1.148) that does not require the supply of reduction equivalents by the THF-type cofactor, they are NADPH oxidases that use flavin adenine nucleotide (FAD) to mediate hydride transfer the genomes of these respective organisms do not specify orthologues of dihydrofolate reductase (see Section 7.17.2.3.2(viii)). ... 

The reaction catalyzed by dihydrofolate reductase appears superficially simple and involves the transfer of a hydride ion from the 4 position of NADH to the 6 position of the substrate, dihydrofolate (Figure 7)... 

Mammalian dihydrofolate reductases can also catalyze the transfer of a hydride ion to the C-7 position of folate in a reaction affording dihydrofolate, thus enabling the utilization of the fully oxidized vitamin from nutritional sources/ ... 

The experimentally observed velocities of the hydride transfer reaction has been explained by tunneling in fact, dihydrofolate reductase is a paradigm enzyme for the study of tunneling processes in biological systems. 

A complete kinetic scheme has been established for the enzyme from both sources. The L. casei dihydrofolate reductase followed a reaction sequence identical to the E. coli enzyme (Scheme I) moreover, none of the rate constants varied by more than 40-fold Figure 20 is a reaction coordinate diagram comparing the steady-state turnover pathway for E. coli and L. casei dihydrofolate reductase, drawn at an arbitrary saturating concentration (1 mM) of NADPH at pH 7. The two main differences are (i) L. casei dihydrofolate reductase binds NADPH more tightly in both binary (E-NH, -2 kcal/mol) and tertiary (E NH-H2F, - 1.4 kcal/mol E-NH-H4F, - 1.8 kcal/mol) complexes, and (ii) the internal equilibrium constant (E-NH H2F E-N-H4F) for hydride transfer is less favorable for the L. casei enzyme (1 kcal/mol). These changes, as noted later, are smaller than those observed for single amino acid substitutions at the active site of either enzyme. Thus, the overall kinetic sequence as well as the... 

The role of protein motions in dihydrofolate reductase (DHFR) has been examined using molecular dynamics simulations as well as experimental studies of mutant enzymes. DHFR catalyzes transfer of hydride from NADPH to dihydrofolate via a tunneling mechanism. Molecular dynamics simulations of complexes of the enzyme with both substrates show both correlated and anticorrelated motions (see Figure 23). " Mutations... 

Andres J, Safont VS, Martins JBL, Beltran A, Moliner V (1995) AMI and PM3 transition structure for the hydride transfer a model of reaction catalyzed by dihydrofolate-reductase. Theochem-J Mol Stmc 330 411 16... 

Andres J, Mohner V, Safont VS, Domingo LR, Richer MT, Krechl J (1996) On transition structures for hydride transfer step a theoretical study of the reaction catalyzed by dihydrofolate reductase enzyme. Bioorg Chem 24(1) 10-18... 

Doron D, Major DT, Kohen A, Thiel W, Wu X (2011) Hybrid quantum and classical simulations of the dihydrofolate reductase catalyzed hydride transfer reaction on an accurate semi-empirical potential energy surface. J Chem Theory Comput 7(10) 3420-3437 Field M (2007) A practical introduction to the simulation of molecular systems, 2nd edn. Cambridge University Press, Cambridge... 

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