Alkylation of Ribonuclease from Aspergillus saitoi with Iodoacetate and Iodoacetamide
1973 Volume 73 Issue 4 Pages 705-716
Details
1973 Volume 73 Issue 4 Pages 705-716
1) In order to obtain the knowledge on the active site of ribonuclease from Aspergillus saitoi (RNase M)[EC 2. 7. 7. 17], chemical modifications of RNase M by various kinds of halo fatty acidsand iodoacetamide were studied.
2) RNase M was inactivated most effectively by bromoacetate and iodoacetate at pH 4.0, and by 3-bromopropionate at pH 8.0 among the halo fatty acids tested. The rateof inactivation by iodoacetamide is higher at pH 8.0 and lower at pH 4.0 than by iodoacetate.
3) The rate of inactivation of RNase M by iodoacetate was maximum at pH 3.5, and about one residue of carboxymethyl group was incorporated into RNase M with concomitant loss of enzymatic activity. The inactivation of RNase M by iodoacetate was protected in the presence of competitive inhibitors such as2'-AMP, indicating possible involvement of the amino acid residue to be carboxymethylated in the active site of the enzyme.
4) Amino acid composition of carboxymethylated RNase M (CM RNase M) indicated that about one residue of carboxymethyl group was introduced into N3-position of histidine. The results were also confirmed by analyses of 14C-CM RNase M.
5) In contrast to iodoacetate, iodoacetamide inactivated RNase M at pH 8.0 with the introduction of three carboxamidomethyl groups into the enzyme. Amino acid composition of carboxamidomethylated RNase M (CAM RNase M) indicated that two moles of carboxamidomethyl groups were introduced into N3-position of histidine residues.
6) Spectrophotometric titration of phenolic groups and fluorescence spectrum of CM RNase M and CAM RNase M indicated that the gross conformation of the modified enzymes is notmuch different from that of the native enzyme. However, the structures of native RNase M and CAM RNase M differ slightly from CM RNase M by judging from the rate of reaction with dinitrofluorobenzene.
7) The binding constants of 2'-AMP with CAM RNase M and CM RNase M were about 1/1.5 and 1/10 as that with the native enzyme, respectively, indicating that CAM RNase M has a gross conformation moresimilar to RNase M than CM RNase M.
