Studies on Adenosine Triphosphate Transphosphorylases X. REACTIVITY AND ANALYSIS OF THE SULFHYDRYL GROUPS OF THE CRYSTALLINE ADENOSINE TRIPHOSPHATE-CREATINE TRANSPHOSPHORYLASE FROM CALF BRAIN

Abstract The native calf brain ATP-creatine transphosphorylase, like its muscle enzyme counterpart (Jacobs, H. K., Keutel, H. J., Yue, R. H., Okabe, K., and Kuby, S. A., Fed. Proc., 27, 640 (1968)) contains 2 reactive sulfhydryl groups per mole, or 1 per polypeptide chain of this dimeric protein (Yue, R. H., Jacobs, H. K., Okabe, K., Keutel, H. J., and Kuby, S. A., Biochemistry, 7, 4291 (1968)), which may be titrated with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). In the presence of denaturants, other sulfhydryl groups are revealed. The kinetics of reactions between DTNB and the sulfhydryl groups of both the calf brain and the muscle isoenzyme have been measured under several sets of denaturing conditions. A comparison of their respective second order velocity constants reveals significant differences between these two isoenzymes, i.e. in the reactivity or accessibility of their types of sulfhydryl groups. Reactions between DTNB and the 2 normally reactive sulfhydryl groups per mole of either isoenzyme leads to essentially complete loss in enzymatic activity, which may be recovered after reduction; therefore, like the rabbit muscle enzyme (Mahowald, T. A., Noltmann, E. A., and Kuby, S. A., J. Biol. Chem., 237, 1535 (1962)), these 2 exposed sulfhydryl groups appear to be essential either for the catalysis or their active conformation, or both. In contrast to the calf muscle isoenzyme, with a total of 8 sulfhydryl groups per mole, or 4 per subunit, there are 10 sulfhydryl groups per mole of brain isoenzyme. These 2 additional sulfhydryl groups per mole of brain isoenzyme are not readily titrated by DTNB even in 8 m urea, 4 m guanidinium chloride, or 1.8% lauryl sulfate; or by p-chloromercuribenzoate under other denaturing conditions. However, under acidic conditions (pH 3.2 at 30°), they may be reacted with 4,4'-dithiodipyridine. A comparison between the muscle isoenzyme and its more acid-labile brain isoenzyme may be made in terms of the kinetics of reaction with 4,4'-dithiodipyridine as a function of pH. Another unique property of the native brain isoenzyme, in contrast to its muscle isoenzyme, appears to be its unusual susceptibility to oxidation. In the presence of atmospheric O2, there is a progressive decay in the 2 normally reactive sulfhydryl groups per mole, both in their number and rates of reaction with DTNB. After prolonged reactions with O2, secondarily reactive, or normally inaccessible, sulfhydryl groups are then exposed for reaction with DTNB in a complex and multiphasic type of reaction. Finally, the mixed disulfide (TNB-protein) formed by reaction between DTNB and the 2 normally reactive sulfhydryl groups per mole, could be prepared from both calf isoenzymes. Although the TNB-muscle protein appeared to be stable between pH 5.5 and pH 8.5, the 5-thio-2-nitrobenzoate group could be released in a pseudo first order fashion from the brain isoenzyme derivative, under mildly acidic conditions, and in the absence of any external reductant. At 30°, ionic strengths above 0.2, the kinetics of 5-thio-2-nitrobenzoate release seemed to be approximately first order (0.85 measured) in H+ over a narrow pH range of 5.5 to 6.5. This H+-initiated process presumably is the result of a pH-dependent unfolding which leads to a thiol-disulfide reaction between this mixed disulfide and a normally unreactive or inaccessible sulfhydryl group within the brain isoenzyme protein.