Kinetics of Chemical Modification of Arginine Residues in Mitochondrial Creatine Kinase from Bovine Heart: Evidence for Negative Cooperativity

L. V. Belousova^1* and E. L. Muizhnek²

¹Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (7-095) 939-3955; E-mail: anton@protein.bio.msu.su

²Moscow Scientific Institute of Medical Ecology, Simferopolsky Bulvar 8, Moscow 113149, Russia

^* To whom correspondence should be addressed.

Received July 9, 2003; Revision received August 26, 2003

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The kinetics of chemical modification of arginine residues in mitochondrial creatine kinase (mit-CK) from beef heart by 4-hydroxy-3-nitrophenylglyoxal (HNPG) have been studied with simultaneous registration of enzyme inactivation. Experiments showed that complete inactivation of mit-CK corresponded to modification of two arginine residues per mit-CK monomer. The data on the modification kinetics can be described by the sum of two exponential terms and suggest strong negative cooperativity in the binding of HNPG to arginine residues. The rate constants for the fast and slow phases of modification differ by a factor of about 50. The corresponding rate constants for inactivation differ by a factor of about 30. The rate constant for the slow stage of inactivation is twice as large as that for the rate constant for the slow stage of modification, i.e., the inactivation process is ahead of the modification process.

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KEY WORDS: mitochondrial creatine kinase, inactivation, modification, essential arginine residues, negative cooperativity

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