STRUCTURE AND FUNCTION OF RIBONUCLEASE-T1 .21. MODIFICATION OF HISTIDINE RESIDUES IN RIBONUCLEASE-T1 WITH IODOACETAMIDE

When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) [Aspergillus oryzal] was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37.degree. C about 90% of the original activity was lost in 24 h. The half-life of the activity was about 8 h. The binding ability for 3''-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of 2 histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5-8 in this reaction. 3''-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. Analyses of the chymotrypic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates; histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. Alkylation of histidine-40 was slowed down most in the presence of 3''-GMP. Apparently histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.