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.
Mozumdar
S
S
Department of Chemistry, State University of New York at Buffalo 14214-2094, USA.
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003619:Dansyl Compounds / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; C059710:8-azido-2'-O-dansyladenosine triphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium