The Amino Acid Sequence Around the “Reactive” Sulfhydryl Groups in Adenosine Triphosphocreatine Phosphotransferase^*

Abstract

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-

Dinitrophenylation and Thiolysis as a Tool in Protein Chemistry

“…Attempts have been made to achieve group specificity in dinitrophenylation by using less reactive reagents (e.g, dinitrobenzenesulfonic acid [5]) or by performing the reaction at a pH at which some of the functional groups are relatively much less reactive [6]. However, in practice it was found that the reactivity of certain functional groups in a protein is so much influenced by their microenvironment that it is often possible to find reaction conditions under which FDNB can be led quite selectively not only to one type of functional group but rather to a unique group within a specific site of the protein [7][8][9][10][11].A few years ago we showed [12] that DNP derivatives of sulfhydryls, imidazole irnino-nitrogens, and phenolic hydroxyIs (but not of Q and e amino groups) can be easily displaced by thiols, thus converting dinitrophenylation into a reversible tech- …”

Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases

“…Amino acid sequences around the reactive cysteine residues of creatine kinase, arginine kinase and lmbricine kinase. The sequence for creatine kinase from rabbit muscle was determined by Thomson et al [3] and Mahowald [28], and that from OX brain by Atherton et al [ 5 ] ; the sequence for arginine kinase was determined by Der Terrossian et al [6] of creatine kinase tryptic peptide whereas its C-terminal sequence can be superimposed on the same portion of arginine kinase tryptic peptide. The deletion of one amino acid (glycine or threonine) next to the proline in creatine kinase peptides is compensated by the addition of one glycine in the C-terminal region.…”

Separation of the Two Non‐Identical Subunits of Lombricine Kinase from Lumbricus terrestris Muscle by Chromatography on Sepharose‐Mercurial

“…At pH 8, the molar absorption value of S-Niph-cysteine in the protein was assumed t o be I I .3 mM-l cm-l at 340 nm according to Mahowald [13]. An aliquot of 1Omg dialyzed enzyme was diluted in I mM EDTA, 0.04M NaHCO, buffer pH 8 to a final volume of I ml (enzyme concentration 125 $ I ) and allowed to equilibrate at 4 "C in a 1-cm quartz cell.…”

“…This reagent was previously used to label the two essential -SH groups of creatine kinase [13,27]. The inhibition studies and the stoichiometry of the reaction of N,ph-F with lombricine kinase provided evidence that this reagent is indeed specific to the essential -SH group of this enzyme.…”