Tunneling and coupled motion in the Escherichia coli dihydrofolate reductase catalysis.

H-transfer was studied in the complex kinetic cascade of dihydrofolate reductase. Intrinsic kinetic isotope effects, their temperature dependence, and other temperature-dependent parameters indicated H-tunneling, but no 1 degrees to 2 degrees coupled motion. The data also suggested environmentally coupled tunneling and commitment to catalysis on pre-steady-state isotope effects.

Synthesis and utility of 14C-labeled nicotinamide cofactors.

A new method for the synthesis of the reduced form of beta-nicotinamide [U-14C]adenine dinucleotide 2(')-phosphate([Ad-14C]NADPH) is presented. The present synthesis results in a radioactive material with a specific activity that is greater than 220 mCi/mmol. This method could easily be adapted for syntheses of 14C-labeled NADH, NADP(+), or any nicotinamide cofactors with radiolabels in other positions. Since these cofactors are so ubiquitous, the use and applications of such labeled material has broad implications. The utility of the labeled cofactor for determination of substrates for nicotinamide-dependent enzymes in the nano- to femtomole scale, in alternative enzymatic assays, and in kinetic isotope effect studies is discussed.

Purification, analysis, and preservation of reduced nicotinamide adenine dinucleotide 2'-phosphate.

Nicotinamide-containing cofactors are ubiquitous in biological systems. Consequently, numerous assays have been developed to study such systems that involve a variety of derivatives and isotopically labeled forms of these cofactors. Often the nicotinamide ring is labeled at the C-4 position which is directly involved in the hydride transfer chemistry catalyzed by nicotinamide-dependent enzymes. A label remote from the reactive center is often also required to follow the course of a reaction or the location of the cofactor. Since the necessary labeling pattern can be as unique as the designed experiment, these cofactors need to be synthesized, analyzed, and, most preferably, preserved. The micro-scale preservation of reduced nicotinamides has long been a challenge due to the inherent lability of the reduced cofactors. Furthermore, it has been found that the reduced 2'-phosphorylated cofactor is even less stable (i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) is more labile than reduced nicotinamide adenine dinucleotide). Presented here are methods that were established to purify nicotinamide cofactors via reverse-phase high-performance liquid chromatography (HPLC) and, most importantly, to stabilize NADPH under optimal conditions for long-term storage. Additionally, an analytical HPLC method which achieves 7-min resolution between oxidized and reduced cofactors was developed. This method also results in over 4-min resolution of these nicotinamide cofactors from various derivatives of folic acid. This analysis affords a new analytical assay for the dihydrofolate reductase-catalyzed reaction and several dehydrogenases involved in folic acid metabolism.