The specific alkylation by iodoacetamide of histidine-12 in the active site of ribonuclease.

نویسندگان

  • R G Fruchter
  • A M Crestfield
چکیده

Alkylation of ribonuclease A by iodoacetamide at pH 5.5 yields two products: inactive 3-carboxamidomethylhistidine12.ribonuclease and fully active S-carboxamidomethylmethionine ribonuclease. No derivative substituted at histidine-119 was detected. This is in marked contrast to the products obtained on alkylation by iodoacetate at pH 5.5. The rate of alkylation of histidine in ribonuclease by iodoacetamide is about 1% that of iodoacetate, but the reaction is nevertheless facilitated since it is 10 to 100 times greater than the rate of alkylation of free histidine. The rate of alkylation of histidine-12 by iodoacetamide reaches a maximum at pH 5 to 5.5 and then decreases as the pH is increased. The rate is depressed by the presence of sodium chloride below about pH 5.5 but is unaffected above this pH. The dependence upon pH of this rate of alkylation of histidine-12 indicated that groups ionizing with apparent pK values of 3.5 to 4.3 and 6.2 f 0.2 are involved in maintaining the reactive form of the enzyme. One explanation of the facilitated reaction is an interaction between the dipole of the amide group of the reagent and a dipole on the protein surface. This explanation is supported by the observation that acetamide inhibits the alkylation of histidine-12 by iodoacetamide. It is consistent with previous interpretations of the facilitated reactions of halo acids at either histidine-12 or at histidine-119 to suggest that it is a dipolar orientation of iodoacetamide by histidine-119 and an adjacent carboxyl group which is responsible for the unexpected specificity of iodoacetamide for histidine-12. The rate of alkylation of methionine in ribonuclease by iodoacetamide at 24” was found to be approximately constant between pH 2.3 and 7.0, and independent of sodium chloride and acetamide concentrations. This fact indicates

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عنوان ژورنال:
  • The Journal of biological chemistry

دوره 242 23  شماره 

صفحات  -

تاریخ انتشار 1967