Biochimica et Biophysica Acta, 32I (I973) 329-335 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 66984 INORGANIC PYROPHOSPHATE-PHOSPHOHYDROLYTIC ACTIVITY IN HUMAN SERUM CATALYTIC PROPERTIES OF THE IONIC SPECIES OF PPi AND MgPPi COMPLEXES MOGENS HORDER The University Department of Clinical Chemistry, •rhus Kommunehospital, 8ooo z{rhus C (Den- mark) (Received March 23rd, 1973) SUMMARY The kinetics of the enzyme-catalyzed hydrolysis of inorganic pyrophosphate (PPi) by human serum has been investigated as a function of the ionic species of PPi and Mg at pH 9.0 (37 °C) : Mg 2+, MgPPi 2 , and PPi 4-. Hyperbolic activity-substrate curves are obtained when MgPPi 2- and PPi a- are used as substrates; the activity-substrate curve for total PPI is sigmoid. On the basis of experiments in which MgPPi 2- and PPi 4- are assumed to be alternative substrates for the same site, a catalytic mechanism is proposed: PPi 4-- is the real substrate; Mg 2+ does not directly modify the binding or the hydrolysis of PPi a-, but leads to a decrease in the activity through formation of MgPPi com- plexes. Neither Mg 2+ nor MgPPi complexes bind to the enzyme. INTROD UCTION In previous experiments on the PPi-phosphohydrolytic activity in dialysed serum from healthy, human individuals the activity on the substrate, PPi, as well as on the modifier, Mg, gave a sigmoidal dependence curve (H0rder, M., unpublished). Sigmoidal activity-substrate and activity-modifier curves are not necessarily indicative of multiple interacting binding sites and eonformational changes in the enzyme molecule during binding of substrate and modifiers 1. Kinetic and binding studies on the specific inorganic pyrophosphate phosphohydrolases (EC 3.6.I.I} from different sources 2-4 have demonstrated that binding of Mg by PPi may lead to non- hyperbolic activity-substrate curves. Enzymes that require metals for catalysis, and that have substrates with chelating properties, seem to display this allosteric-like behaviour 5. The present work was undertaken to study the PPi-phosphohydrolytic activity