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A number of sulfhydryl reagents react in tained in over 50% yield. This indicates that stoichiometric proportions to inactivate adenosine both reactive sulfhydryl groups have the same triphosphocreatine (ATP-creatine) phosphotransferase.amino acid sequence throughout these eight Two sulfhydryl groups of the six available on the amino acids. The sequence about these two reacenzyme are preferentially attacked by these reagents.tive sulfhydryl groups is Val-Leu-Thr-CySH-Pro-Ser-Two equivalents of 2,4-dinitrofluorobenzene haveAspNH2-Leu. These data leave no doubt that the been used to label the two reactive sulfhydryl groups potent inhibition of the enzymatic activity of ATPon ATP-creatine phosphotransferase.creatine phosphotransferase caused by the "Sanger'sAfter pepsin digestion at 2°, an octapeptide reagent" (2,4-dinitrofluorobenzene) is owing to its recontaining S-dinitrophenylcysteine residue was ob-action with two sulfhydryl groups.xXfter reaction of the two reactive sulfhydryl groups in ATP-creatine phosphotransferase with FDNB1 (Sanger's reagent), iodoacetic acid, or p-mercuribenzenesulfonate (Kuby and Mahowald, 1959; Mahowald and Kuby, 1960; Mahowald et al., 1962b;Watts et al., 1961), enzymatic activity is completely lost. This inactivation was most effective with FDNB which, when allowed to react at 0°, caused all enzymatic activity to disappear within a few minutes.In this study FDNB was used to label the two reactive sulfhydryl groups for the purpose of isolating a peptide containing the S-DNP-cysteine residue and of determining the sequence of amino acids about these reactive cysteine residues. Thomson et al. ( 1964) have recently reported a 25-amino acid peptide obtained from this enzyme with trypsin as the hydrolyzing enzyme and I4C-labeled iodoacetic acid as the labeling group. The octapeptide obtained in this study with pepsin as the hydrolyzing enzyme corresponds to the portion around the cysteine residue of the peptide isolated by him.Experimental Procedure ATP-creatine phosphotransferase was isolated from rabbit muscle by procedure B of Kuby et al. (1954), and twice recrystallized as previously reported (Maho-