Kinetics Of Chymotrypsin Catalysis

Superior Essays
CHYMOTRYPSIN CATALYSIS
Sarkis Hamalian
ABSTRACT
To examine the enzyme kinetics of chymotrypsin during the catalysis of the synthetic substrate p-nitrophenyl acetate (NPA). Initially, observe the magnitude of the initial burst with regards to the enzyme concentration. Secondly, the analysis of the steady state deacylation rate with regards to the pH of the reaction mixture. Chymotrypsin catalysis is a process that proceeds with two steps, an initial fast step involving the covalent modification of the enzyme to the substrate and the subsequent slower deacylation step which returns the enzyme to its free state (rate determining step). These steps are highly dependent on enzyme concentration and the pH of the environment in which the reaction
…show more content…
The focus of this experiment will be chymotrypsin which falls under the category of serine proteases. Chymotrypsin has been discovered as a digestive enzyme found in pancreatic secretions of animals, whose main function is said to be hydrolysis of peptides that enter the duodenum of the small intestine. As can be determined from its enzyme class, it has an active serine residue which is used to hydrolyze aromatic amino acids such as Phenylalanine, Tyrosine or Tryptophan at their C-terminus. The hydrophobic property of the enzyme pocket (1 per enzyme) is what attracts the aromatic amino acids for cleavage. The catalytic strategies used by chymotrypsin are covalent and acid-base catalysis which are supported by the catalytic triad of serine (covalent catalysis), Histidine and Asparagine (acid-base catalysis). The chymotrypsin catalysis is carried out through two steps the first and faster step being the nucleophilic attack of the carbonyl group at the peptide bond by the serine residue resulting in a covalent modification. The stabilized enzyme intermediate undergoes the slow but essential second step in which water is used to release the covalent bond and regenerate the enzyme. In this case, burst kinetics is examined where the rate limiting step occurs …show more content…
In this case, we were able to prove that chymotrypsin works ideally at pH range between 7 and 8. As can be seen from graph 3 the rate clearly undergoes a rapid rise around pH 7 and continues to the max at pH 8. This increase in reaction rate can be accredited to the specific qualities of the acyl-enzyme intermediate and the reaction environment required to allow the steady state deacylation to proceed. This can be explained by examining the catalytic triad of chymotrypsin, mentioned earlier, and its importance in the activity of the enzyme. In particular we can examine the histidine residue of the triad, which in the second step is responsible for being protonated by water which ultimately results in the release of acetate from the serine residue resulting in a free enzyme that is able to react with a new substrate. The significance lies in the pKa of the Histidine side chain which is 6.8. So when the pH of the reaction mixture is above this pKa, ideally pH 7-8, the side chain can be protonated to allow the steady state deacylation step to occur

Related Documents

  • Improved Essays

    Despite the ambiguity in deprotonation of a serine residue, Zn(II) ion at M2 position then stimulates Ser102 to attack the phosphorus center of the substrate to initiate the leaving of an alcohol group and to form a phosphoseryl intermediate. This intermediate resembles E-Pi, an enzyme-phosphate complex, in Figure 2 and…

    • 2173 Words
    • 9 Pages
    Improved Essays
  • Improved Essays

    Alkaline Hydrolysis Essay

    • 979 Words
    • 4 Pages

    It is defined as the percentage of broken peptide bonds in relation to the original protein. The degree of hydrolysis achieved in the hydrolysis is determined by the conditions used in the process such as substrate concentration, enzyme / substrate ratio, incubation time as well as the physicochemical conditions such as pH and temperature. Moreover, another factor that will determine the degree of hydrolysis is the nature of the enzyme, characterized by its specific activity and type of activity. Thus, the nature of the enzyme used will not only influence the degree of hydrolysis, but also in the type of peptides produced (Wang et al.,…

    • 979 Words
    • 4 Pages
    Improved Essays
  • Superior Essays

    Enzymology and Catalytic Mechanism Carbohydrate Metabolism, Adenosine Triphosphate Western Governors University ENZYMES • Are proteins that act as catalysts, and carry out chemical reactions.…

    • 1053 Words
    • 5 Pages
    Superior Essays
  • Great Essays

    v = d[P]/(dt )= Vmax[S]/((KM + [S])) Equation (a): Henri Michaelis-Menten HMM equation Firstly; Michaelis–Menten equation (1) describe the enzymatic reactions rate , the concentration present as (S ) substrate , maximum rate ( ) by the system, The Michaelis constant is the substrate concentration . E+S □(↔┴k ) ES □(→┴kb ) E+P E.q. (b): enzymatic reactions rate at which the product in the reaction Secondly; By this mathematical model, the enzyme (E) binding to a (s) substrate to form ES which will convert to product finally , the , , and represent the rate constants, the double arrows show a reversible process of theenzyme-substrate…

    • 1581 Words
    • 7 Pages
    Great Essays
  • Improved Essays

    Krebs Cycle Metabolism

    • 1426 Words
    • 6 Pages

    One things importants, the citric acid cycle is an amphibolic pathway. The krebs cycle a role in anabolism. For example the Anaplerotic reaction, these reactions replenish the intermediates of the citric cycle that are removed to serve as biosynthetic precursors. Thry are regulated to keep the level to intermerdiates high enough to support the activity of the citric cycle. And for finally we will talk about the glyoxylate cycle , the glyoxylate cycle catalyzes the net conversion of acetate to succinate.…

    • 1426 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    According to the Michaelis-Menten model, an enzyme-substrate complex (ES) is formed when an enzyme (E) binds to a substrate (S). After the ES has formed it can then be utilized to form the required product. When the degree of an enzymatic reaction is measured it is done so at different substrate concentrations (the degree of an enzymatic reaction is also known as the velocity). This is an indication that the rate of the reaction largely hinges on the concentration of the substrate. When the substrate level is low, the reaction is in the first order, meaning that the substrate concentration can influence the velocity.…

    • 1035 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    Then, Rct is affected by both the property of electrode and modification of the electrode surface [19]. The enzyme loading on the electrode surface could be verified by comparison of the Rct values [20]. Because the identical sets of electrodes were used for the EIS measurements, hydrolysate is the strong influential factor towards the charge transfer. The hydrolysate liquid after the DAP of lignocellulosic biomass involves various undefined materials derived from the biomass by hydrolysis and pyrolysis.…

    • 1213 Words
    • 5 Pages
    Improved Essays
  • Great Essays

    Here we will discuss about Claisen ester condensation reaction. 1. Claisen ester condensation – Here we are taking specific example of Ethyl acetate (CH3COOET). When ethyl acetate is treated with strong base like –OET and then the reactants are refluxed then they produces a β-keto ester via several steps. In this case also the last step is most important because it helps to drive the equilibrium of every step in the forward direction.…

    • 1316 Words
    • 6 Pages
    Great Essays
  • Improved Essays

    When oxygen is not present, fermentation of the pyruvic molecule will occur. When oxygen is present acetyl coA enters into the Krebs/ Citric Acid Cycle inside the mitochondrial matrix and is oxidized to CO2 while reducing NAD to NADH. With each turn of the Krebs cycle, a pyruvate is used and one turn of the cycle produces 3NADHs, 1 ATP, 1 FADH2 and CO2. The Krebs cycle also produces water when NADH and FADH2 shuttle electrons down the electron transport chain to oxygen, which is the last electron acceptor; while at the same time protons are being pumped from the mitochondrial matrix to the intermembrane space allowing oxygen to be reduced to water. NADH and FADH2 can be used in the electron transport chain to create ATP by oxidative phosphorylation.…

    • 1178 Words
    • 5 Pages
    Improved Essays
  • Superior Essays

    Analysis Of Trypsin-1

    • 1406 Words
    • 6 Pages

    This increased electronegativity allows the histidine to attract the hydrogen in the serine’s –OH group. This boosts the nucleophilic behaviour of the serine and allows it to attack and facilitate the cleavage of the substrate. A calcium cofactor for this reaction is bound by residues 75, 77, 80 and 85 [3] (Appendix 2). Trypsin-1 is created in the pancreas and excreted extracellularly [3] (Appendix 2). It then enters the small intestine during digestion, where it facilitates the breakdown of proteins for their subsequent absorption into the blood stream [6].…

    • 1406 Words
    • 6 Pages
    Superior Essays