Question

2. hypothesize the best conditions (pH and temperature) under which the enzyme chymotrypsin functions with an appropriate reference. also, hypothesize what will occur in the presence of an inhibitor and the type of inhibition.

EXPERIMENT 5: ENZYME ACTIVITY WITH a-CHYMOTRYPSIN Prelab Assignment 1. Prepare a flow chart, covering one half of the experimental work (either part A and C if your lab bench is on the window side of the lab or part B and D if your locker is on the door side of the lab). 2. Hypothesize the best conditions (pH and Temperature) under which the enzyme chymotrypsin functions with an appropriate reference. Also, hypothesize what will occur in the presence of an inhibitor and the type of inhibition. 3. Prepare proper tables to collect data from parts A-D. This can be computer generated if you wish but MUST be in your lab notebook. 4. Bring the following to lab: lab coat: safety glasses or safety goggles; closed toed shoes; ruler; sharple marker; pencil; pen; calculator; lab notebook. Theory An enzyme is a biological catalyst that can increase the rate of a reaction without being, consumed itself. Enzymes play a large role in a lot of biochemical reactions. For example, the enzyme pepsin begins to digest protein molecules while they are in our stomach. Yet, a majority of the digestion of proteins occurs once the food enters the small intestines. Here enzymes like trypsin, chymotrypsin, elastase, and carboxypeptidase convert protein molecules into amino acids, dipeptides, and tripeptides. The dipeptides and tripeptides are converted to amino acids by other enzymes and then the free amino acids move throughout our body. One of the enzymes mentioned, chymotrypsin (CT), is an endopeptidase, a major pancreatic proteolytic enzyme with a molecular weight of 21,600. CT's catalytic sites contain a highly reactive serine residue that confers CTs preference for peptide bonds on the carboxyl side of the aromatic side chains of L-phenylalanine, L-tryptophan and L- tyrosine. CT also cleaves peptide bonds of some large hydrophobic residues like L- methionine. CT is studied in this experiment because of its ability to react with GPNA (N-glutaryl-L-phenylalanine-p-nitroanilide), a colourless compound. The product, P- nitroaniline, absorbs strongly at wavelength of 410nm and this absorbance can be related to the activity of the enzyme. The ability of enzymes to function depends on a variety of factors like the presence of inhibitors, pH and temperature. Simple enzyme reactions involve the enzyme (E), the substrate ($) and the product (P). An enzyme-substrate (ES) intermediate and sometimes, an inhibitor (I) are also a part of the reactions. The general equation is: E+S ES E +P and E+I EI (competitive inhibitors only)

Answer 1

The best conditions under which the enzyme chymotrypsin functions are

i) pH = 7.8

ii) Temperature = 50 ^oC

$\alpha$-Chymotrypsin is completely inhibited by 10 mM Cu²⁺ and Hg²⁺.

The typical range for the Michaelis-Menten constant of Chymotrypsin is K_M = 15 mM