Interaction of Zn2+ and Eu3+ with bovine liver glutamate dehydrogenase.
Bovine liver glutamate dehydrogenase is potently inhibited by Zn2+ ions. At pH 7.0 a kinetic dissociation constant for Zn2+ of 18 microM is obtained. The fluorescent lanthanide Eu3+ competes for the Zn2+-binding site and relieves the Zn2+-induced inhibition, but does not cause inhibition. Studies on the effects of Zn2+ or Eu3+ on the tertiary and quaternary structure of the enzyme by the use of protein fluorescence, heat-stability and re-activation after guanidinium chloride denaturation indicate that, whereas Zn2+ affects both tertiary and quaternary structure, Eu3+ does not affect either, consistent with its lack of effect on enzymic properties. Eu3+ fluorescence had a strong excitation peak at 395 nm with emission at 456 nm. In the presence of glutamate dehydrogenase the fluorescence emission is shifted to 501 nm. Eu3+, with high-affinity binding site and distinctive fluorescence properties after binding, would appear to be an ideal fluorophore for use in conformational studies or resonance-energy-transfer studies.
Bell
E T
ET
Department of Biochemistry, University of Rochester Medical Center, NY 14642.
Stilwell
A M
AM
Bell
J E
JE
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005063:Europium / Q000494:pharmacology*; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000494:pharmacology*
D005063:Europium; D005969:Glutamate Dehydrogenase; D015032:Zinc