A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction.
A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector 'kinetic connectivities' between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H(2)O(2). To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I-adrenaline complex.
Vrzheshch
Peter V
PV
International Biotechnological Center, Lomonosov Moscow State University, Leninskiye Gory, Laboratory Building B, Room 610, Moscow 119992, Russian Federation. peter@genebee.msu.ru
Batanova
Elena A
EA
Mevkh
Alevtina T
AT
Varfolomeev
Sergei D
SD
Gazaryan
Irina G
IG
Thorneley
Roger N F
RN
D000818:Animals; D002384:Catalysis; D004837:Epinephrine / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D006861:Hydrogen Peroxide; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D004837:Epinephrine