Characterization of tightly bound substrates in pure preparations of dihydrofolate reductase: implications for studies on enzymes.
Investigations have been made to determine the identity and binding characteristics of the pterins that are bound tightly to dihydrofolate reductases which are isolated from vertebrate sources by a well established procedure. This procedure involves the binding of enzyme to a Sepharose-methotrexate column, elution with dihydrofolate and removal of free dihydrofolate by dialysis or by size-exclusion chromatography. Addition to such preparations of NADPH results in oxidation of the nucleotide and from the progress curves so obtained, it is possible to identify the bound pterin and to calculate the stoichiometry of binding. The data indicated that stoichiometric amounts of dihydrofolate or folate plus dihydrofolate were bound to the dihydrofolate reductases. It can also be concluded that binding occurs at the active sites of the enzymes. Enzyme preparations from which bound pterin had been removed by isoelectric focussing reacted with folate or dihydrofolate to form 1:1 enzyme-pterin complexes from which the pterin could not be removed by dialysis or by size-exclusion chromatography. From the magnitude of the dissociation constants for the enzyme-pterin complexes and the concentration of enzyme present in fractions after the step involving affinity chromatography on Sepharose-methotrexate, it could be concluded that the presence of bound folate and/or dihydrofolate in pure preparations of dihydrofolate reductase is simply a consequence of an association-dissociation reaction. Preparations of the enzyme from bacterial sources were also found to contain bound pterins. The findings have implications with respect to kinetic and thermodynamic studies on dihydrofolate reductase and other enzymes which are isolated by affinity chromatography techniques.
Williams
E A
EA
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Morrison
J F
JF
D002845:Chromatography; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
C010920:dihydrofolate; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase