Escherichia coli serine hydroxymethyltransferase is a 94-kDa homodimer. Each subunit contains a covalently attached pyridoxal-P, which is required for catalytic activity. At which step pyridoxal-P binds in the folding pathway of E. coli serine hydroxymethyltransferase is addressed in this study. E. coli serine hydroxymethyl-transferase is rapidly unfolded to an apparent random coil in 8 M urea. Removal of the urea initiates a complete refolding to the native holoenzyme in less than 10 min at 30 degrees C. Several intermediates on the folding pathway have been identified. The most important information was obtained during folding studies at 4 degrees C. At this temperature, the far-UV circular dichroism spectrum and the fluorescence spectrum of the 3 tryptophan residues become characteristic of the native apoenzyme in less than 10 min. Size exclusion chromatography shows that under these conditions the refolding enzyme is a mixture of monomeric and dimeric species. Continued incubation at 4 degrees C for 60 min results in the formation of only a dimeric species. Neither the monomer nor dimer formed at 4 degrees C bind pyridoxal phosphate. Raising the temperature to 30 degrees C results in the formation of a dimeric enzyme which rapidly binds pyridoxal phosphate forming active enzyme. These studies support the interpretation that pyridoxal phosphate binds only at the end of the folding pathway to dimeric apoenzyme and plays no significant role in the folding mechanism.

A new rapid procedure for purifying 10-formyltetrahydrofolate dehydrogenase results in 90 mg of pure enzyme from two rabbit livers. This abundant liver enzyme is known to bind its product tetrahydropteroylpentaglutamate (H4PteGlu5) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H4PteGlu5. There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H4PteGlun, suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme. H4PteGlu5 complex. How this tightly bound H4PteGlun is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C1-tetrahydrofolate synthase, which use H4PteGlu5 as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H4-PteGlu5 has a dissociation constant near 15 nM which is 60-fold lower than the Ks for 10-formyl-H4PteGlu5. Fluorescence titration studies also yield a Kd of about 20 nM for H4PteGlu5. Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase not only abolishes product inhibition but also increases the initial rate of its activity by about 2-fold. Passage of a reaction mixture of 10-formyltetrahydrofolate dehydrogenase down a size exclusion column results in enzyme with 1 equiv of H4PteGlu5 bound per subunit. However, addition of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase results in a rapid transfer of this bound folate to these enzymes. Evidence is also presented that the tightly bound folate is in equilibrium with solvent H4PteGlu5.

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An experiment for the synthesis of N-acyl derivatives of natural amino acids has been developed as part of the Distributed Drug Discovery (D3) program. Students use solid-phase synthesis techniques to complete a three-step, combinatorial synthesis of six products, which are analyzed using LC−MS and NMR spectroscopy. This protocol is suitable for introductory organic laboratory students and has been successfully implemented at multiple academic sites internationally. Accompanying prelab activities introduce students to SciFinder and to medicinal chemistry design principles. Pairing of these activities with the laboratory work provides students an authentic and cohesive research project experience.