Kinetics of Chymotrypsin

Topics: Amino acid, Enzyme, Protein Pages: 19 (3558 words) Published: March 10, 2014
The Kinetics of α-Chymotrypsin

Introduction

Chymotrypsin is a protease which cleaves proteins by a hydrolysis reaction, it does this by adding a molecule of water to a peptide bond. Although the hydrolysis reaction is thermodynamically favoured in the absence of a catalyst the half-life for a typical hydrolysis reaction by a protease is between 10 and 100 years, needless to say it is extremely slow1. Though this is true peptide bonds are hydrolysed within milliseconds in the body in the presence of catalysts. The kinetic stability of chymotrypsin which gives it hydrolysis resistance is due to the resonance structure that accounts for the planarity of the peptide bond. Such is the strength of this resonance structure that it confers partial double bond character. The carbonyl carbon atom in peptides is less electrophilic and therefore less susceptible to nucleophilic attack than carbonyl carbon atoms in most other compounds. This means that for a protease to function it has to cleave peptide-bonds at an unreactive carbonyl group1.

For this experiment we looked at the kinetics of chymotrypsin. We calculated the extinction co-efficient of p-nitrophenoxide which was important for the enzyme kinetic assays we carried out later in the experiment. We then measured the concentration of some stock solutions of chymotrypsin using both the Folin and BIORAD assays so we could get an idea of which assay is more accurate when measuring enzyme concentration. Finally we looked at the kinetics of chymotrypsin by carrying out various enzyme kinetic assays. We used different enzyme and substrate concentrations in our assay so we could determine the mechanism of action of chymotrypsin.

Chymotrypsin breaks down proteins in the digestive systems of mammals as well as other organisms. It selectively cleaves peptide bonds on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine and methionine1. Chymotrypsin operates by covalent catalysis whereby the serine residue acts as a nucleophile to attack the unreactive carbonyl carbon atom of the substrate so that it becomes briefly attached.

The kinetics of chymotrypsin action can be monitored by measuring the absorbance of a coloured product when the enzyme is acting on a substrate analog. In this experiment the coloured product is p-nitrophenoxide and the substrate is p-nitrophenyl trimethylacetate. Under steady state conditions, the reaction is known to favour Michaelis-Menten kinetics with a KM of 20µM and a kcat of 77s-1. The hydrolysis begins with an initial rapid burst phase followed by a steady state phase1.

The two phases of the reaction are caused because a covalently bound enzyme-substrate intermediate is formed. The acyl group initially becomes covalently attached to the enzyme as p-nitrophenoxide is released. This acyl-enzyme intermediate is then hydrolysed to release the carboxylic acid component of the substrate and regenerate the free enzyme1. This means that one molecule of p-nitrophenoxide is produced rapidly from each enzyme molecule as the acyl-enzyme intermediate is formed. Both phases are needed for enzyme turnover however it takes a lot longer to effectively free the enzyme for use by the hydrolysis of the acyl-enzyme intermediate1.

Chymotrypsin is spherical and is made up of 3 polypeptide chains conjoined by disulphide bonds. It is synthesized as chymotrypsinogen which is a single polypeptide which yields 3 chains after it is activated by proteolytic cleavage. Serine 195 marks the active site of chymotrypsin and lies in a cleft on the surface of the enzyme1. The unusually high reactivity of serine 195 is determined by the structure of the active site. There is a hydrogen bond between the imidazole ring of histidine 57 and the side chain of serine 195. The NH group of the imidazole ring is also hydrogen bonded to the carboxylate group of aspartate 102. The three aforementioned residues together make up...

References: 1. Berg, J., Tymoczko, J., and Stryer, L. (2012) Biochemistry, 7th edition, New York: W. H. Freeman and Company.
2. Bender et al. (1967) Journal of Chemical Education 44, 84-88.
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