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Reactivity of tyrosine in bovine pancreatic deoxyribonuclease with p-nitrobenzenesulfonyl fluoride.

1982; Elsevier BV; Volume: 257; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(19)83825-5

1083-351X

T H Liao, Ruby Ting, Jennifer Yeung,

Biochemical and Molecular Research

A protein modifying agent, p-nitrobenzenesulfonyl fluoride, has been shown to be specific for tyrosine.The reaction conditions are mild (in aqueous buffers at 25 "C), and the rate increases with pH.The reaction product is a derivative of 0-sulfonylated tyrosine which yields 0-(p-nitrobenzenesulfonyl) tyrosine (NBS-Tyr) after acid hydrolysis.The extent of modification can be measured as NBS-Tyr by high performance liquid chromatography (HPLC).The reagent was used to study the reactivity and the importance in enzymic function of tyrosine in bovine pancreatic DNase in either the Ca2+ form or the Ca2+free form.Ca2+-DNase is inactivated faster than Ca2+free DNase; at 50% inactivation, the number of residues of NBS-Tyr was approximately 1 in Ca2+-DNase and 2.5 in Ca2+-free DNase.Chromatographic analysis of tryptic-chymotryptic hydrolysates on Sephadex G-25 and by HPLC also showed differences in patterns for the two forms modified by the reagent.Peptides containing NBS-Tyr were identified by comparing absorbances at 254 nm and 280 nm and isolated by using Sephadex G-25 and HPLC.Their locations in the primary structure were assigned based on amino acid composition.Because each modified peptide appeared as a distinct peak on analysis by HPLC, the extent of modification at each tyrosine site was readily determined.Results are summarized as follows.(a) In Ca2+-free DNase, Tyr-62 reacts about 20 times faster than N-acetyltyrosine (N-Ac-Tyr), but its modification does not affect enzymic activity; the same residue in Ca2+-DNase reacts at the same rate as AT-Ac-Tyr.(b) In Ca2+-free DNase the residue crucial to enzymic activity is Tyr-145 which reacts slightly faster than N-Ac-Tyr.(c) In Ca2+-DNase, while "145 reacts at a lower rate than the free amino acid, Tyr-172 reacts 4.6 times faster than N-Ac-Tyr, and the sum of Tyr-145 and Tyr-172 accounts for loss of activity, indicating that Tyr-172 is also crucial in DNase.(d) T w o other residues (Tyr-51 and Tyr-73) are only slightly modified in Ca2+-DNase but are significantly modified in Caz+-free DNase, These two modifications in Ca2+free DNase do not seem to account for loss of activity; however, reaction at one site appears to influence that at the other, suggesting proximity of the two in the three-dimensional structure.Bovine pancreatic DNase requires Mg2+ or Mn2+ for activity (1).In studying the effect of other divalent metal ions, Wilberg