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Divergent Enzyme Kinetics and Structural Properties of the Two Human Mitochondrial Creatine Kinase Isoenzymes

Divergent Enzyme Kinetics and Structural Properties of the Two Human Mitochondrial Creatine... Abstract The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p 4 symmetry with lattice parameters of 145 Å, indicating a ‘flattening’ of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism ( K d > K m ) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower K m for creatine (1.01mM) and ATP (0.11mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Chemistry de Gruyter

Divergent Enzyme Kinetics and Structural Properties of the Two Human Mitochondrial Creatine Kinase Isoenzymes

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References (45)

Publisher
de Gruyter
Copyright
Copyright © 2000 by the
ISSN
1431-6730
DOI
10.1515/BC.2000.131
pmid
11154064
Publisher site
See Article on Publisher Site

Abstract

Abstract The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p 4 symmetry with lattice parameters of 145 Å, indicating a ‘flattening’ of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism ( K d > K m ) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower K m for creatine (1.01mM) and ATP (0.11mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).

Journal

Biological Chemistryde Gruyter

Published: Nov 15, 2000

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