J. Biol. Chem., Vol. 267, Issue 4, 2173-2178, Feb, 1992
The active site of creatine kinase. Affinity labeling of cysteine 282 with N-(2,3-epoxypropyl)-N-amidinoglycine
DD Buechter, KF Medzihradszky, AL Burlingame and GL Kenyon
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143.
Epoxycreatine (N-(2,3-epoxypropyl)-N-amidinoglycine) is an affinity label
of creatine kinase that irreversibly and completely inactivates the enzyme
(Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 254, 1879-1886).
To identify active site residues of rabbit muscle creatine kinase, the site
of modification of it by epoxycreatine has been determined. Separation by
high performance liquid chromatography of a tryptic digest of
[14C]epoxycreatine-modified creatine kinase yielded two radiolabeled
peptides. The larger of these consisted of amino acids Ala-266 through
Arg-291 and was labeled with epoxycreatine at Cys-282. Attempts to purify
completely the other labeled peptide were not successful; however, it was
possible to obtain, by tandem mass spectrometry, a collision-induced
dissociation spectrum of it from a mixture of several peptides. This
peptide was a fragment (amino acids Val-279 through Arg-291) of the
previously identified peptide and was also labeled at Cys-282. Model
studies with cysteine and epoxycreatine have demonstrated that opening of
the oxirane ring occurs by attack of the cysteine thiolate at the terminal
carbon of the epoxide. These results are consistent with previous studies
on the base lability of the label; however, a carboxyl group in the active
site is not labeled, as had been previously suggested. These results
provide evidence that Cys-282 is located in or near the creatine-binding
site and will also be important in identifying and delineating the
boundaries of the active site of creatine kinase.
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