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Carboxymethylation of a minor ribonuclease from Aspergillus saitoi
Abstract:
(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.
Insights
Iodoacetate inactivates RNase Ms by modifying a glutamic acid residue in the active site. This modification affects nucleotide binding and reactivity, suggesting its crucial role in enzyme function.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Ribonucleases (RNases) are crucial enzymes in RNA metabolism.
- Understanding the active site residues of RNases is key to elucidating their catalytic mechanisms.
- RNase Ms is a specific enzyme whose active site has not been fully characterized.
Purpose of the Study:
- To identify the specific amino acid residue in RNase Ms modified by iodoacetate.
- To investigate the functional consequences of this modification on enzyme activity and substrate binding.
- To determine the role of the modified residue in the catalytic mechanism of RNase Ms.
Main Methods:
- Enzyme inactivation studies using iodoacetate at varying pH and denaturant concentrations.
- Competitive inhibition assays with various nucleotides.
- Chemical modification of RNase Ms with iodoacetate and subsequent analysis of incorporated groups.
- Radiolabeling and enzymatic digestion to identify the modified amino acid.
- Spectroscopic analysis (CD spectrum) and reactivity assays (dinitrofluorobenzene) to assess conformational changes.
Main Results:
- RNase Ms inactivation by iodoacetate was optimal at pH 6.0 and inhibited by denaturants.
- Nucleotide inhibitors protected RNase Ms from inactivation, with varying efficacy.
- Iodoacetate modification resulted in the incorporation of one carboxymethyl group, identified as being on a glutamic acid residue.
- Carboxymethylated RNase Ms (CM RNase Ms) showed altered binding affinities for certain nucleotides.
- Conformational analysis indicated subtle differences between native and CM RNase Ms.
Conclusions:
- A specific glutamic acid residue in RNase Ms is essential for its catalytic activity.
- Modification of this glutamic acid residue by carboxymethylation impacts nucleotide binding.
- These findings highlight the critical role of this glutamic acid residue in the active site of RNase Ms.