Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

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Abstract
The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal RNase was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent Cys-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted definition pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The superactivity of intersubunit disulfides of seminal RNase is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal RNase Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the 2 Cys residues of interest, was even more reactive. The superreactivity at neutral pH of Cys residues at positions 31 and 32 of bovine seminal RNase is primarily dependent on the nearby presence of positively charged groups, particularly the .epsilon.-NH2 of Lys-34, and is influenced by the adjacency of the 2 thiols and by the protein tertiary structure.