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‐[α‐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 μM 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 μM 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.