Lennie Cocco

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase

/sup 13/C NMR spectra of Streptococcus faecium dihydrofolate reductase containing (/sup 13/C-guanidino) arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppM. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that /sup 13/C NMR of labeled protein can be used in studies of protein-ligand interactions. An example of the use of /sup 13/C-depleted material is also presented.

13C NMR spectra of organoaluminum compounds: evidence for an oxygenaluminum π-interaction

Abstract The 13C NMR spectra of some trialkylaluminum compounds and a series of alkoxy-bridged and amido-bridged organoaluminum compounds are reported and discussed. In the alkoxy-bridged compounds the OC resonance is sensitive to substitution at the aluminum atom and the degree of oligomerization. In the amido-bridged compounds the analogous carbon (NC) resonance shows no variation upon changes of substituents at the aluminum. These observations are used as evidence for the existence of an oxygen to aluminum pπdπ dative bond in the alkoxy-bridged compounds.

Molecular Basis for the Interaction of Polyglutamates of Folic Acid and its Analogs with Dihydrofolate Reductase

Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes. The polyglutamates do not bind more tightly than folate to the S. faecium reductase under any conditions examined, but bind somewhat more tightly than folate to the L. casei reductase at low pH (less than 7) and to the bovine liver enzyme at pH 7-9. Increasing concentrations of KC1 decrease the binding of all three ligands to the L. casei and bovine liver enzymes. Increasing pH markedly raises the dissociation constants for all complexes of the L. casei reductase, but has only slight effects on the complexes of the S. faecium reductase. Complexes of the bovine enzyme are affected to an intermediate degree by pH, but the folate complex is affected much more than those of the polyglutamates. Model building studies have been performed with a three-dimensional model of the complex of L. casei reductase with NADPH and methotrexate. Additional glutamyl groups were added in gamma-linkage to the glutamate moiety of the complexed methotrexate. A proposed mode of binding of the pteroyl polyglutamates is discussed and sequence comparisons are used to predict residues that might be involved in polyglutamate binding by reductase from other sources.