Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding.
The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, 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 two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The Km value of pantothenase to pantothenate 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 7.6mM in phosphate buffer at 20 degrees C; the corresponding Kapp.m value at pH7 is 15 mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km rofile. pK is 7.0 in phosphate buffer at 20 degrees C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were--14kJ/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 K1 values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K1 being 0.36mM, and the corresponding Kapp.m about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.
D000581:Amidohydrolases / Q000378:metabolism*; D001665:Binding Sites; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010070:Oxalates / Q000494:pharmacology; D010071:Oxaloacetates / Q000494:pharmacology; D010205:Pantothenic Acid / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013696:Temperature
D002021:Buffers; D010070:Oxalates; D010071:Oxaloacetates; D010205:Pantothenic Acid; D010664:Phenylmethylsulfonyl Fluoride; D000581:Amidohydrolases