Dihydrofolate dehydrogenase

Pasquali, P., Landi, L., Caldarera, C. M. and Marchetti, M. Effects of orotic acid on dihydrofolate dehydrogenase and on tetrahydrofolate-dependent enzymes in the chick liver. Biochim. Biophys. Acta, 158, 482-484 (1968)... 

The latter method, called the PI-FEP/UM approach, allows accurate primary and secondary kinetic isotope effects to be computed for enzymatic reactions. These methods are illustrated by applications to three enzyme systems, namely, the proton abstraction and reprotonation process catalyzed by alanine race-mase, the enhanced nuclear quantum effects in nitroalkane oxidase catalysis, and the temperature (in)dependence of the wild-type and the M42W/G121V double mutant of dihydrofolate dehydrogenase. These examples show that incorporation of quantum mechanical effects is essential for enzyme kinetics simulations and that the methods discussed in this chapter offer a great opportunity to more accurately model the mechanism and free energies of enzymatic reactions. 

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). 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

This enzyme [EC 1.5.1.3], also called tetrahydrofolate dehydrogenase, catalyzes the reversible reaction of 7,8-dihydrofolate with NADPH to produce 5,6,7,8-tetrahy-drofolate and NADP+. The enzyme isolated from mammals and some microorganisms can also slowly catalyze... 

The enzymatic activity of amido phosphoribosyltransferase (P-Rib-PP— PR A) is low and flux through the de novo pathway in vivo is regulated by the end-products, AMP, IMP and GMP. Inhibition of reaction 1 by dihydrofolate polyglutamates would signal the unavailability of /V1()-formyl tetrahydrofolate, required as a substrate at reactions 3 and 9 of the pathway. The purine pathway is subject to further regulation at the branch point from IMP XMP is a potent inhibitor of IMP cyclohydrolase (FAICAR—> IMP), AMP inhibits adenylosuccinate synthetase (IMP—> sAMP) and GMP inhibits IMP dehydrogenase (IMP— XMP). 

Other classes include aldehyde dehydrogenase inhibitors aldose reductase inhibitors aminopeptidase inhibitors aromatase inhibitors carboxypeptidase inhibitors cyclooxygenase inhibitors dihydrofolate... 

Reaction of 1 with ATP in presence of Mg and kinase gives 2-amino-4-hy-droxy-6-hydroxymethyldihydropteridine pyrophosphate (2, DHP pyrophosphate). The latter is converted into 7,8-dihydropteroate (3, DHP) by its reaction with p-ami-nobenzoic acid (PABA) and DHP synthetase. Addition of L-glutamic acid to DHP in the presence of the enzyme dihydrofolate synthetase (DHF synthetase) yields 7,8-di-hydrofolate (4, DHF), which undergoes reduction by the enzyme dihydrofolate reductase to afford 5,6,7,8-tetrahydrofolate (5, THF). DHF may also lose a hydrogen molecule in the presence of DHF dehydrogenase to form folic acid (6). 

---