The chemical reactivity of the thiol group in the active centre of ficin

Abstract

• 1.

  1. The irreversible inhibition of ficin (EC 3.4.4.12) by chloroacetamide or iodoacetamide is due to the alkylation of the thiol group of one particular cysteine residue in the enzyme.

• 2.

  2. The variation of the rate of alkylation of ficin with pH shows that the reactive thiol group of ficin behaves essenitally as a freely ionising roup, pK_a′ = 8.55 (25 °; I, 0.1). Hence it is concluded that this group is unlikely to exist in a hydrogen-bonded form in the free enzyme.

• 3.

  3. The rates of reaction of ficin with chloroacetamide or iodacetamide were accelerated in the presence of substrates or products of catalytic activity without the stoichiometry of the overall reaction being affected. The rate of addition of the essential thiol of ficin to N-ethylmaleimide was unaffected by substrate or substrate analogues.

• 4.

  4. The nucleophilicity of the essential thiol group of ficin in S_N² reactions was found to be greater than that of simple thiol compounds whereas in the addition reaction to N-ethylmaleimide the converse was found.

• 5.

  5. A possible reason for the high nucleophilicyt of ficin in S_N² reactions and the acceleration caused by the presence of substrate or substrate analogues is given.