The velocity of the pantothenase [EC 3.5.1.22]-catalyzed hydrolysis of pantothenate was studied over pH 5.5-9 and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing 2 ionizations of the enzyme, with 1 ionizable group located at the substrate-binding site and the other at the inhibitor-binding site. The Km value of pantothenase to pantotenate depends on the buffer used and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6 mM in phosphate buffer at 20.degree. C; the corresponding .**GRAPHIC**. value at pH7 is 15 mM. The pK value of the ionizable group at the substrate-binding site was measured by 2-methods: from the pH-rate profile and from the pH-Km profile. pK is 7.0 in phosphate buffer at 20.degree. C, ranging in various buffers between 6.9-7.3. The van''t Hoff enthalpies of substrate binding and H+ ion binding were-14 kJ/mol and -50 kJ/mol, respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the Ki values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent Ki being 0.36 mM, and the corresponding .**GRAPHIC**. about 1.8 mM at pH 7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.
