Differentiation of substrate-binding sites in pyruvate kinase by selective photoaffinity labeling

The four substrate-binding sites in porcine liver pyruvate kinase have been labeled with the photoaffinity reagent 8-azido-2'-O-dansyl-[alpha-32P]ATP (AD-ATP) under different experimental conditions. In the dark, the native pyruvate kinase was reversibly and competitively inhibited by AD-ATP, with KI = 2.8 microM and KADP = 0.18 mM. Under UV-irradiation, the enzyme was covalently labeled in the presence of Mg2+ by AD-ATP and inactivated irreversibly. Measurement of this photoinactivation process in the presence of various concentrations of ADP gave KI = 4.0 microM and KADP = 0.2 mM. A linear plot of the relative specific activity of the partially photolabeled enzyme after gel-filtration vs. the number of label per pyruvate kinase molecule introduced in the presence of Mg2+ shows that each covalent label completely inactivates a tetrameric pyruvate kinase molecule. In the strict absence of Mg2+, up to three substrate-binding sites in each pyruvate kinase molecule can be labeled by AD-ATP without decreasing enzyme activity. Subsequent addition of Mg2+ enables AD-ATP to label the remaining site and inactivate the enzyme. These observations show that there are one catalytic and three non-catalytic substrate-binding sites in each pyruvate kinase molecule. A probable structural reason for possible functional differentiation of intrinsically identical substrate-binding sites in all tetrameric enzymes is suggested.