Kinetics of the creatine kinase reaction in neonatal rabbit heart: an empirical analysis of the rate equation.
Here we define the kinetics of the creatine kinase (CK) reaction in an intact mammalian heart containing the full range of CK isoenzymes. Previously derived kinetic constants [Schimerlik, M. I., & Cleland, W. W. (1973) J. Biol. Chem. 248, 8418-8423] were refit for the reaction occurring at 37 degrees C. Steady-state metabolite concentrations from 31P NMR and standard biochemical techniques were determined. 31P magnetization transfer data were obtained to determine unidirectional creatine kinase fluxes in hearts with differing total creatine contents and differing mitochondrial CK activities during KCl arrest and isovolumic work for both the forward reaction (MgATP synthesis) and reverse reaction (phosphocreatine synthesis). The NMR kinetic data and substrate concentration data were used in conjunction with a kinetic model based on MM-CK in solution to determine the applicability of the solution-based kinetic models to the CK kinetics of the intact heart. Our results indicated that no single set of rate equation constants could describe both the KCl-arrested and working hearts. We used our experimental data to constrain the solution-derived kinetic model and derived a second set of rate equation constants, which describe the isovolumic work state. Analysis of our results indicates that the CK reaction is rate limited in the direction of ATP synthesis, the size of the guanidino substrate pool drives the measured CK flux in the intact heart, and during isovolumic work the CK reaction operates under saturating conditions; that is, the substrate concentrations are at least 2-fold greater than the Km or Kim for each substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
McAuliffe
J J
JJ
Department of Anesthesia and Pediatrics, University of Cincinnati College of Medicine, Ohio 45267-0531.
Perry
S B
SB
Brooks
E E
EE
Ingwall
J S
JS
D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism*; D005583:Fourier Analysis; D007527:Isoenzymes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D011817:Rabbits
D007527:Isoenzymes; D003402:Creatine Kinase