Hydride-transfer reactions dihydrofolate reductases

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... 

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. 

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... 

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). 

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.

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. 

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). 

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/ ... 

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... 

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... 

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