THE JOURNAL OF Bromcrc~~ CHEMISTRY Vol. 254, No. 6, Issue of March 25, pp. 1879-1886, 1979 Prmted LIZ U.S.A. Affinity Labeling of Creatine Kinase by IV-(2,3-Epoxypropyl)-N- amidinoglycine * (Received for publication, August 14, 1978) Michael A. MarlettaS and George L. Kenyong From the Departments of Pharmaceutical Chemistry and Biochemistry and Biophysics and the Cardiovascular Research Institute, University of California, San Francisco, California 94143 The compound N-(2,3-epoxypropyl)-N-amidinogly- tine (epoxycreatine) was synthesized to probe the ac- tive site of rabbit muscle creatine kinase (EC 2.7.3.2) because of its structural similarity to the normal sub- strate creatine and because of the reactivity of its epox- ide moiety toward nucleophiles. In the presence of epoxycreatine and in the absence of other substrates, complete inhibition of enzymatic activity is observed which does not return after exhaustive dialysis. The inhibition follows saturation kinetics. At 0°C (pH 7.45), Iii”act is 335 mM compared to a KS value of 24 mM for creatine at 30°C. The inactivation half-time at saturat- ing epoxycreatine concentration is 4.2 min. In a com- petitive manner, creatine protects the enzyme from inactivation by epoxycreatine. Product studies indicate that epoxycreatine is also a substrate for the enzymatic reaction in the presence of both M$+ and ATP. The partitioning of epoxycreatine between phosphorylation by MgATP and inactivation of the enzyme was followed spectrophotometrically at 25°C and found to be 15 turn- overs/inactivating event. The inactivation half-time in the presence of MgATP is slightly greater than that when MgADP is present, which also is slightly greater than that of epoxycreatine alone. The so-called transition state analogue complex of MgADP2- l NOs- l creatine provides strong protection against inactiva- tion by epoxycreatine. The stoichiometry of the reac- tion, determined with 14C-labeled epoxycreatine, is one site modified per subunit of enzyme, even in the pres- ence of excess reagent. These results indicate that epox- ycreatine is binding at the active site and that it is therefore an affinity label. Creatine kinase (adenosine 5’-triphosphate:creatine N- phosphotransferase, EC 2.7.3.2) catalyzes the reversible trans- fer of a phosphoryl group from ATP to creatine. The enzyme, both soluble and membrane-bound, is found in abundant supply in both muscle and brain tissues and is generally considered to be important in maintaining constant ATP * This research was supported by Grant AM 17323 from the Na- tional Institute of Arthritis, Metabolism and Digestive Diseases. A portion of this work was presented at the 61st Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Ill., April, 1977 (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ National Institutes of Health Predoctoral Trainee, 1974 to 1977 (Training Grant GM 00728 to the Department of Pharmaceutical Chemistry). Present address, Department of Chemistry, Massachu- setts Institute of Technology, Cambridge, Mass. 02139. - 6 Recipient of United States Public Health Service Research Ca- reer Devklopment Award AM 00014 (1975 to 1980). levels in such tissues (2, 3). Creatine kinase consists of two apparently identical subunits with the molecular weight of the dimer being 82,000. It requires a divalent metal ion for catalysis. In this paper, we have examined the rabbit skeletal muscle enzyme exclusively. Chemical modifications of creatine kinase from rabbit skel- etal muscle have focused mainly on the reactive sulfhydryl group, and a variety of reagents are known to block that functionality (4-13). Other studies have involved modification of a lysine residue (9, 14-16), an arginine residue (17, 18), and a histidine residue (19). Creatine kinase must rank as one of the most thoroughly studied enzymes with respect to both reversible and irreversible inhibition (20). Until the present work (1)) however, none of the irreversible inhibitors has been an active-site-directed substrate analogue (affinity label). Our efforts to synthesize an affinity label structurally re- lated to creatine were stimulated by the lack of information concerning the active site of creatine kinase. The complete primary amino acid sequence is not yet known; in fact, the only such structural information that has been reported is a sequence containing 25 amino acids around the reactive sulfhydryl group (21,22). An affinity label, owing to its specific interaction with the enzyme, should begin to fill a large gap in our knowledge of the active site of this enzyme that is so important in the bioenergetics of muscle action. Moreover, the ability to inactivate creatine kinase with high selectivity in vivo should permit detailed investigations concerning the bioenergetics of ATP utilization in muscle action without the complicating features of the ATP s phosphocreatine inter- conversion (23). The design of the creatine-based affinity label, epoxycrea- tine,’ resulted from detailed investigations of the bulk toler- ances of the active site of creatine kinase. Preliminary studies had shown that both glycocyamine (24) and N-ethylglycocy- amine (25) could act as substrates. HzN\~ $ ‘.. _I’ Y NH2 /T c /R co*e where: R = H, glycocyamine; CHB, creatine; CHsCHs, N-ethylglycocyamine; CH2CH&H3, N-propylglycocyamine; CH:, CH-CH2, epoxycreatine. ‘0’ More detailed and quantitative studies revealed the extent ’ The abbreviations used are: epoxycreatine, N-(2,3-epoxypropyl)- N-amidinoglycine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid; HPLC, high performance liquid chromatography. 1879 by guest on September 22, 2016 http://www.jbc.org/ Downloaded from