Laurence T. Plante

[23] Pteroyllysine-agarose in the purification of dihydrofolate reductase

Publisher Summary Dihydrofolate reductase from antifolate-resistant Lactobacillus casei can be isolated in homogeneous form and in excellent yield by the affinity column chromatographic method described in this chapter. This method differs from other currently available affinity column procedures for purification of dihydrofolate reductase in using the ligand pteroyllysine, a folate analog that is a substrate rather than an inhibitor, and which has only moderate affinity for the enzyme. Other affinity columns for dihydrofolate reductase use ligands which are inhibitors, and most often the ligand chosen has been methotrexate, which binds extremely tightly to the enzyme (K i -9 M). The advantages of the described system are that enzyme elution is achieved easily under very mild conditions, thus allowing quantitative recovery of activity, and that loss of small amounts of ligand from the column during chromatography does not interfere with subsequent enzyme studies.

Interaction of 13C-enriched folate with dihydrofolate reductase studied by carbon magnetic resonance spectroscopy

Abstract In the presence of dihydrofolate reductase the carbon magnetic resonance spectrum of folate labeled at the benzoylcarbonyl carbon with 13 C contains two broadened peaks arising from free and enzyme-bound folate, the latter appearing over 2 ppm upfield from free folate. Addition of TPN + causes sharpening of both peaks indicating formation of a single folate-TPN + -enzyme ternary complex. Methotrexate specifically displaces folate from the ternary complex regenerating a single sharp resonance at 170.4 ppm characteristic of free folate. Line width changes show that folate is bound more tightly in the ternary than in the binary complex. Increased shielding of this carbonyl upon binding is inconsistent with its participation in a H-bond.

[69] Preparation of folic acid specifically labeled with carbon-13 in the benzoyl carbonyl

Publisher Summary This chapter discusses the preparation of folic acid, specifically labeled with Carbon-13, in the benzoyl carbonyl. The use of folic acid and its reduced forms, labeled with radioactive and stable isotopes, has contributed greatly to understanding its roles in enzymology. Improved nuclear magnetic resonance (NMR) technology for carbon-13 has also provided the means for obtaining additional information. Such studies require 13 C-labeled ligands and the method discussed in this chapter describes the synthesis of folic acid labeled with 13 C in its benzoyl carbonyl. In this method, the acetylpteridine aldehyde is condensed with p -aminobenzoic acid- 13 C-carboxyl in acetic acid to give, after reduction with dimethylamine borane, a quantitative yield of N 2 -acetylpteroic acid. The N 10 -amino group is blocked with trifluoroacetic anhydride. The N 2 -acetyl-N 10 -trifluoroacetylpteroic acid is converted to the mixed anhydride, with isobutyl chloroformate, and the reaction, with diethyl glutamate, gives, after hydrolysis, folic acid- 13 C-benzoyl carbonyl in about 70% overall yield. The folic acid- 13 C-benzoyl carbonyl shows the same ultraviolet absorption spectra as authentic folic acid and the same mobility in three aqueous solvent systems; the chemical shift of the labeled carbonyl was observed at 170.4 ppm (relative to tetramethylsilane). The labeled folic acid is reduced quantitatively by nicotinamide adenine dinucleotide phosphate (NADP)H in the presence of a catalytic amount of L. casei dihydrofolate reductase.

Nα-Ipteroyltetra (γ-Glutamyl)]-Lysine as a Ligand for the Purification of Thymidylate Synthetase by Affinity Chromatography

Abstract Nα[Pteroyltetra (γ-glutamyl)]-lysine Sepharose was synthesized and shown to be a stable, high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.

Interaction of the carboxamide of NADPH with Lactobacillus casei dihydrofolate reductase

Abstract Dihydrofolate reductase from Lactobacillus casei and its complexes with NADPH and methotrexate yield well-resolved Raman spectra. The 1685-cm−1 Raman band assigned to the carboxamide of NADPH persists in the NADPH-enzyme binary complex but is absent from the NADPH-methotrexate-enzyme ternary complex. This is ascribed to stabilization of the polarized form of the carboxamide by H bonding to the NH and CO groups of Ala 6 and Ile 13 of the peptide backbone.

[70] Preparation of aminopterin and p-aminobenzoylglutamic acid specifically labeled with carbon-13 in the benzoyl carbonyl

Publisher Summary This chapter discusses the preparation of aminopterin and p -aminobenzoylglutamic acid specifically labeled with carbon-13 in the benzoyl carbonyl. Aminopterin (4-amino-4-deoxypteroylglutamic acid) is the representative of a large group of folic acid antagonists, the best known of which is methotrexate (N 10 -methyl derivative). These compounds are strong inhibitors of the enzyme dihydrofolate reductase and are useful in cancer chemotherapy. Aminopterin labeled with 13 C in the benzoyl carbonyl has been prepared for the study of enzyme ligand interactions by carbon magnetic resonance spectroscopy. The intermediate p -aminobenzoylglutamic acid- 13 C-benzoyl carbonyl is also useful in this regard. In the method described in this chapter for the preparation of aminopterin and p -aminobenzoylglutamic acid, the p -aminobenzoic acid- 13 C-carboxyl is acylated with trifluoroacetic anhydride and condensed with diethyl glutamate, by the mixed anhydride procedure to give, after hydrolysis, p -aminobenzoylglutamic acid- 13 C-benzoyl carbonyl. This compound is reacted with 2,4-diamino-6-bromomethylpteridine hydrobromide by the procedure of Piper and Montgomery to give aminopterin- 13 C-benzoyl carbonyl. The p -aminobenzoylglutamic acid- 13 C-benzoyl carbonyl and aminopterin- 13 C-benzoyl carbonyl obtained show ultraviolet spectra and paper chromatographic behavior identical to authentic specimens. The 13 C nuclear magnetic resonance (NMR) spectra of the two compounds show chemical shifts for the labeled carbonyl of 170.4 and 170.6 ppm (relative to TMS), respectively.