What is lysine found in

Epoxycreatine N- 2, 3-epoxypropyl ; -N-amidino-1987 ; . There are two known cytosolic creatine kinase subglycine ; is an affinity label of creatine kinase that units: brain B ; and muscle M ; , each of molecular weight irreversiblyand completely inactivates the enzyme approximately 43, 000. These associate to form the dimeric Marletta, M. A., and Kenyon, G. L. 1979 ; J . Biol. muscle MM ; , brain BB ; , and heterodimer MB ; isozymes. Chem. 254, 1879-1886 ; . To identify active site resiIn addition to the cytosolic isozymes, there is also an isozyme dues of rabbit muscle creatine kinase, the site mod- associated with the inner mitochondrial membrane. Creatine of ification of it by epoxycreatine has been determined. kinasecDNA from a number of species, including rabbit Separation by high performance liquid chromatogra- muscle Putney et al., 1984 ; , hasbeen cloned and sequenced phy of a tryptic digestof [`4C]epoxycreatine-modified Babbitt et al., 1986; Chen et al., 1991 ; . creatine kinaseyielded two radiolabeled peptides. The The considerable amount of information available regardlarger of these consisted of amino acids Ala-266 ing the kinetic and mechanistic aspects of the reaction catathrough Arg-291 and was labeled with epoxycreatine a t Cys-282. Attempts to purify completely the other lyzed by creatine kinase has been reviewed Kenyon andReed, labeled peptide were not successful; however, it was 1983 ; . Attempts to delineate the roles played by individual have a amino acid residuesin catalysishave beennumerous and possible to obtain, by tandem mass spectrometry, resulted in the identification of several residues that may be collision-induced dissociation spectrum of it from a mixture of several peptides. This peptide was a frag- involved in catalysis and or binding. Chemical modification ment amino acids Val-279 through Arg-291 ; of the and NMRspectroscopy studies have implicatedarginine Bor1975; James, 1976 ; , lysine Jamesand previously identified peptide and was also labeled at dersandRiordan, Cys-282. Model studies with cysteine and epoxycrea- Cohn, 1974; Kassab et al., 1968 ; , and histidine Clarke and tine have demonstrated that opening of the oxirane Price, 1979; Cook et al., 1981; Rosevear et al., 1981 ; residues. ring occurs by attack of the cysteine thiolate at the The arginine and lysine side chains are proposed to interact terminal carbon of the epoxide. These results are con- electrostatically with the phosphate groups of the nucleotide, sistent with previous studies the base lability the and the histidineresidue is believed to act as a general acid on of label; however, a carboxyl group in the active site is base to protonate deprotonate the guanidinium nitrogen that not labeled, as had been previously suggested. These is phosphorylated. A carboxyl group, which must be ionized results provide evidence that Cys-282 is located in or for the binding of both creatine and phosphocreatine, has near the creatine-binding site and will also impor- been implicated from pH rate studies Cook et al., 1981 ; . None be tant in identifying and delineating the boundaries of of the above groups has been located with any certainty in the active site creatine kinase. of the primary sequence of the enzyme. Creatine kinase also possesses one highly reactive sulfhydryl per subunit. This group can be modified with a number Creatine kinase ATP: creatine N-phosphotransferase, EC of sulfhydryl-specific reagents Kenyon and Reed, 1983; Ma2.7.3.2 ; catalyzes the reversible transfer of a phosphoryl group howald et ul., 1962; O'Sullivan, 1971 ; , often, but not always, with complete loss of activity der Terrossian and Kassab, from MgATP to creatine, leading to phosphocreatine and 1974 ; . EPR spectroscopy studie: of a MgADP. Creatine kinase playscritical role in cellular energy 1976; Smith and Kenyon, metabolism Saks et al., 1975; Seraydarian and Abbott, 1976 ; enzyme spin-labeled at this sulfhydryl place it within 10 A of and in the transport and utilization of "high energy phos- the metal-binding site Cohn al., 1971; Taylor et al., 1969 ; . phate"withinthe cell Jacobus, 1985 ; . Inadditiontoits This residue has been identified as Cys-282 in the primary physiological role, creatine kinase is important in the diag- sequence of rabbit muscle creatine kinase Mahowald, 1965; nosis of several diseasestates, including myocardialinfarction Thomson et al., 1964 ; and is conserved in all known creatine Wu, 1989 ; and muscular dystrophy GruemerandPrior, kinase sequences. The role that Cys-282 plays in catalysis is not known. * This work was supported by United States Public Health Service Epoxycreatine N- 2, 3-epoxypropyl ; -N-amidinoglycine ; ' Grant AR 17323 to G. L. and National Science Foundation Grant Scheme 1, ; is a creatine-based affinity label of creatine DIR 8700766 and United States Public Health Service Grant 1979 ; . In a process that follows RR01614 to A. L. The costs of publication of this article were kinase Marletta and Kenyon.

Amino acid lysine titration curve