CHROMIUM(III) MODIFICATION OF THE FIRST METAL-BINDING SITE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) is activated b y Cr2+ as the sole activator under anaerobic conditions. PEPCK was mod ified with Cr3+. Starting with either Cr2+ or Cr3+ Cr3+ has the distin ct advantage of being a paramagnetic cation that could serve as a para magnetic probe. Activators Mn2+, Mg2+, and Co2+ protect against Cr3+ i ncorporation. 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 +(n(1))-PEPCK forms enzyme-substrate complexes similar to those observ ed for the Mn2+(n(1))-PEPCK complex (n(1) represents the metal ''enzym e binding site'' as opposed to the metal ''nucleotide binding site''). Cr3+(n(1))-PEPCK requires an additional divalent cation for activity, an indication of two metal sites on PEPCK. Cr3+(n(1))-PEPCK retains 1 5% residual activity as compared to unmodified PEPCK and demonstrates normal Michaelis-Menten kinetics. This is the first report of an activ e Cr3+-modified enzyme complex.