Enzyme Inhibition
Ribonuclease A and Tosylphenylalanine Chloromethyl Ketone(TPCK)
Read about the goals of Ribonuclease A,TPCK and enzyme inhibition in modern protein chemistry.
Ribonuclease A: Enzyme Inhibition
Bovine pancreatic ribonuclease A (RNase A) has been the most significant goal of the modern protein chemistry for the past 50 years.It is an endonuclease enzyme-made up of 124 amino acid residues- which cleaves specifically at the 3`-end of pyrimidine end of nucleosides. A large number of RNase A studies have been continuously carried out to determine structure and function relationships in the enzyme and recently; an efficient expression system using E.coli has been developed, making it possible to produce mutant enzymes on a large scale. Chemical modification as well as genetic manipulation has been useful to substitute not only polar but non-polar amino acid residues. It is evident from early X-ray diffraction analyses; that two of the histidine residues, His12 and His119, function as catalytic residues. In addition recombinant DNA techniques also show that His 12 and His 119 operate as an acid and a base and that the involvement of the protonated His119 to catalysis is not constrained to the protonation of an oxygen atom of the substrate leaving group. Moreover, Chatani & Hayashi (2001) show that His119 participates directly in transition state stabilization via hydrogen bonding.
A slightest deviation of His119 causes a drastic decrease in catalysis making the arrangement of RNase very strict because it is determined by its surrounding residues. Some residues, which are located in the vicinity of the catalytic residues, contribute to the catalytic reactions by interacting with catalytic residues, in order to achieve a proper position or pKa
Consequently, a chemical modification or mutation of His 119 to Gly will affect not only the folding rate but also the thermal stability of RNase A; resulting in a loss of most of RNase activity.
TPCK: Enzyme Inhibition
Tosylphenylalanine chloromethyl ketone (TPCK) is a reagent that binds to chymotrypsin in a substrate-like manner, but, instead of being hydrolyzed, it alkylates a single histidine residue in the active site of the enzyme. This selective chemical modification is termed ‘affinity labelling’.
Chymotrypsin and trypsin are affliates of a family of enzymes known as the serine proteases. Chymotrypsin and Trypsin are created in the pancreas and excreted into the intestine. They are made as pre-enzymes and stimulated in the intestine by partial proteolytic degradation. Their amino-acid sequences are very similar with 62 out of 257 amino acids being alike. Likewise their 3-D structures are very comparable – small alpha-helix - and a beta sheet in the centre
However Chymotrypsin is a protease that cleaves peptide bonds on the carboxyl side of aromatic or large hydrophobic amino acids. Alternatively, trypsin shows specificity for peptide bonds in which the carbonyl carbon is from positively charged amino acids such as arginine or lysine. Consequently, these two enzymes hydrolyze similar bond in peptides but they have diverse target amino acids.
Chymotrypsin - Phe Tyr Trp
Trypsin - Arg Lys
When an analogous approach is used in developing an affinity label for trypsin, the resulting reagent, tosyl-lysine chloromethyl ketone (TLCK), like TPCK, selectively alkylates the active site histidine residue. Therefore, TLCK does not inhibit chymotrypsin and, conversely, TPCK does not inhibit trypsin. Thus, these two reagents can be distinguished between the activities of two enzymes based on their primary specificity.
TPCK is not an effective inhibitor of trypsin however, reagents specific for trypsin can be produced by replacing the phenylalanine residue of TPCK with arginine or lysine.
Resources:
Berg, J.M., Tymoczko, J.L., Stryer, L. (2002).Supplements Supporting Biochemistry, Fifth Edition; 357-433.
Chantani, E., Hayashi, R. (2001). Functional and Structural Roles of Constituent Amino AcidsResidues of Bovine Pancreatic Ribonuclease A. Journal of Bioscience and Bioengineering 92(2):98-107.
Wilbur H. Campbell, (1995);Enzyme Mechanisms - Serine Proteases Part II.
