Semisynthetic Ribonuclease A
2-D Semisynthetic RNase A
Introduction
The synthesis of a fully active semi-synthetic RNase A supports the
hypothesis that the amino acid sequence of a protein solely dictates
the formation of an active enzyme and demonstrates that an enzyme with
the catalytic activity and specificity of a naturally produced enzyme can
be made in laboratory. Semisynthetic RNase A illustrates that functional
enzymes can be produced from merely the individual constituent amino acid
residues. Polypeptide synthesis can provide new routes to the study of
enzymes through the selective modification of natural proteins to
assay individual roles of amino acids in folding and catalysis.
Function
The structure to function relationship is clearly exhibited by semisynthetic
RNase A. In the RNase A protein, the removal of six C terminal residues,
leaving ,
completely halts enzymatic activity (Martin, 1987). However, a complex of
RNase 1-118 with a synthetic polypeptide comprising the
restores enzymatic activity to RNase A. Upon the addition of the synthetic
chain, the semisynthetic enzyme adopts a structure that closely resembles
that of natural RNase (Martin, 1987). The restoration of the structure
reconstitutes the enzymatic activity of RNase to 98% (Martin, 1987).
Synthetic Method
The RNase 1-118 was prepared by successive digestion of RNase A pepsin and
carboxypeptidase A (Doscher, 1983). The synthetic component, RNase 111-124,
was prepared by the use of solid-phase peptide synthetic mothods, in which
the peptide chain was assembled in the stepwise mannar while it was attached
at one end to a solid support. The peptide chain was extented by repetitive
steps of deprotection, neutralization and coupling until the desired sequence
was obtained (Lin, 1970). It was important that the synthesis proceeds rapidly
and in high yields to prevent side reactions or by-products.
Related Web-links
1. Introduction to Ribonuclease A by Raines:
http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf
2. Solid Phase Synthesis by Merrifield (Nobel Prize Winner):
http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf
3. Chemical Synthesis of Proteins:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez
4. Refined Crystal Structure: http://www.ncbi.nlm.nih.gov/pubmed/3680234
References
Martin, Philip D., Marilynn S. Doscher, and Brian F. P. Edwards. "The Redefined
Crystal Structure of a Fully Active Semisynthetic Ribonuclease at 1.8-A Resolution."
The Journal of Biological Chemistry
262.33 (1987): 15930-5938.
Marilynn S. Doscher, Philip D. Martin and Brian F.P. Edwards, "Characerization
of the Histidine Proton Nuclear Magnetic Resonance of a Semisynthetic Ribonuclease."
Biochemistry, 1983,22,4125-4131.
Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731.
David J. Boerema, Valentina. A. T., Stephen B. H. Kent, "Total Synthesis by
Modern chemical Ligation Methods and High Resolution (1.1-A) X-ray structure
of Ribonuclease A. Biopolymers. 2008;90(3):278-86.
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