Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26.
The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent and which differ in the orientation of the pteridine ring of the substrate in the binding site. The Asp26----Asn mutant of L. casei dihydrofolate reductase has been prepared by oligonucleotide-directed mutagenesis and studied by one- and two-dimensional 1H-NMR spectroscopy. NMR studies of the mutant enzyme--folate--NADP+ complex show that this exists to greater than 90% in a single conformation over the pH* range 5-7.1. The single conformation observed corresponds to conformation I (the 'methotrexate-like' conformation) of the wild-type enzyme--folate--NADP+ complex. These observations demonstrate that Asp26 is the ionizable group controlling the pH-dependence of the conformational equilibrium seen in the wild-type enzyme.
Jimenez
M A
MA
Department of Biochemistry, University of Leicester, UK.
Arnold
J R
JR
Andrews
J
J
Thomas
J A
JA
Roberts
G C
GC
Birdsall
B
B
Feeney
J
J
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D001224:Aspartic Acid; D001216:Asparagine; D013762:Tetrahydrofolate Dehydrogenase