The Tyrosyl Residues at the Active Site of Staphylococcal Nuclease

Abstract

Abstract The reaction of the extracellular nuclease of Staphylococcus aureus with tetranitromethane yields an inactive enzyme containing 5 nitrotyrosyl residues. If nitration is performed in the presence of deoxythymidine 3',5'-diphosphate and Ca++, enzymatic activity is preserved and only 2 or 3 tyrosyl residues are nitrated. Reaction of the nuclease with a small molar excess of tetranitromethane results in selective nitration of the tyrosine residue at position 85. This mononitrotyrosyl nuclease can be purified on phosphorylated cellulose columns and is catalytically inactive. When the reaction of nuclease with small amounts of tetranitromethane is carried out in the presence of deoxythymidine 3',5'-diphosphate and Ca++ very little nitration of tyrosine 85 occurs, and instead the tryosyl residue at position 115 is selectively nitrated. Tyrosine 115 is unreactive to nitration in the native enzyme, even with a large excess of tetranitromethane. The mononitrotyrosyl 115 nuclease can also be purified on phosphorylated cellulose. It has full deoxyribonuclease activity, but has lost half of the native ribonuclease activity. Nitration of tyrosine 115 causes tyrosine 85 to lose its specific reactivity to nitration, although this residue remains exposed to solvent and is still nitratable. This effect is probably due to an alteration in some specific interaction between these 2 tyrosine residues rather than to a structural change in the protein. The 2 tyrosyl residues thus appear to be spatially adjacent. Judged principally from spectral studies, tyrosine 115 in the native enzyme appears to be in a hydrophobic region where it is inaccessible to nitration. Binding of substrate appears to cause exposure of this residue to an aqueous environment since its ultraviolet spectrum and pK are normalized, and since it becomes accessible to nitration. These results suggest that a conformational change at the active site may result upon binding of deoxythymidine 3',5'-diphosphate to nuclease. In addition to tyrosyl residues 85 and 115, the one at position 27 may also be in the active site, since its nitration is specifically inhibited by deoxythymidine 3',5'-diphosphate and Ca++. The possible functional role of this residue could not be determined, since it could not be selectively nitrated. The 2 unreactive or buried tyrosyl residues of nuclease are probably those at positions 91 and 93, and nitration of these, after protection by reversible acetylation of the other 5 tyrosine residues, does not appear to affect enzymatic activity significantly. The 2 mononitrotyrosyl (115 and 85) derivatives can easily be reduced with sodium dithionite to the corresponding aminotyrosyl derivatives. The latter, owing to the low pK value (4.7) of their aromatic amino groups, can be used for further selective chemical substitutions, such as with the fluorescent dye, dimethylaminonaphthalenesulfonyl chloride, or with p-iodobenzenesulfonyl chloride. The single tryptophanyl residue of nuclease reacts with tetranitromethane to form a yellow derivative, provided this ordinarily buried residue is exposed to the reagent by guanidine hydrochloride or urea.