Steady-state kinetic analysis of substrate pair cycling between two enzymes: application to a mediated electron transport between the cytoplasmic membrane and the periplasmic nitrite reductase of Paracoccus denitrificans.
An extended kinetic model is presented for the process catalysed by two enzymes mutually connected by the cycling of two reversibly interconvertible chemically relative species. Expressions are derived for the steady-state velocity, limiting velocity (V) and the half-saturation concentration of the cycling substrate (A(0.5)). It is shown that the velocity depends on the total concentration of cycling substrate hyperbolically if both enzymes have equal activities. Based on these theoretical considerations, an experimental comparison was made between pseudoazurin and cytochrome c(550) as physiological electron transfer mediators for nitrite reduction in an in vitro reconstituted part of the respiratory chain of Paracoccus denitrificans. Pseudoazurin exhibited 1.7-fold higher V and 14-fold higher A(0.5) than cytochrome c(550) under the experimental conditions used (20 mM Tris chloride, pH 7.3, 30 degrees C).
Kucera
Igor
I
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlárská 2, CZ-61137 Brno, Czech Republic. ikucera@chemi.muni.cz
Kunák
Michal
M
D000465:Algorithms; D001400:Azurin / Q000031:analogs & derivatives* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003574:Cytochrome c Group / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009572:Nitrite Reductases / Q000737:chemistry / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology*; D019897:Periplasm / Q000201:enzymology*; D015219:Substrate Cycling; D019802:Succinic Acid / Q000378:metabolism
D003574:Cytochrome c Group; D009573:Nitrites; C047163:pseudoazurin; D001400:Azurin; C031732:cytochrome C-550; D019802:Succinic Acid; D009572:Nitrite Reductases