Chromium(III) modification of the first metal binding site of phosphoenolpyruvate carboxykinase.
Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) is activated by Cr2+ as the sole activator under anaerobic conditions. PEPCK was modified with Cr3+, starting with either Cr2+ or Cr3+. Cr3+ has the distinct advantage of being a paramagnetic cation that could serve as a paramagnetic probe. Activators Mn2+, Mg2+, and Co2+ protect against Cr3+ incorporation. EPR, CD, and fluorescence studies indicate that Cr3+ was incorporated into the cation binding site of PEPCK. The water proton relaxation rate (PRR) and fluorescence binding studies showed that Cr3+(n1)-PEPCK forms enzyme-substrate complexes similar to those observed for the Mn2+(n1)-PEPCK complex (n1 represents the metal "enzyme binding site" as opposed to the metal "nucleotide binding site"). Cr3+(n1)-PEPCK requires an additional divalent cation for activity, an indication of two metal sites on PEPCK. Cr3+(n1)-PEPCK retains 15% residual activity as compared to unmodified PEPCK and demonstrates normal Michaelis-Menten kinetics. This is the first report of an active Cr3+-modified enzyme complex.
Hlavaty
J J
JJ
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Nowak
T
T
D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D002645:Chickens; D002857:Chromium / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011522:Protons; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry
D002413:Cations, Divalent; D046911:Macromolecular Substances; D011522:Protons; D002857:Chromium; D008345:Manganese; D010729:Phosphoenolpyruvate Carboxykinase (GTP)