Enzymes kinetics.docx

Enzyme is a biological catalyst, and most enzymes are proteins. Contains structures such as: Substrate Active site- where substrate bind. ESC- when substrate binds to active site. Many enzymes have co-factors. Such as: Metal ions such as Fe2+ and Cu+ Coenzymes/ prosthetic groups such as FAD In s...

Enzyme is a biological catalyst, and most enzymes are proteins. Contains structures such as: Substrate Active site- where substrate bind. ESC- when substrate binds to active site. Many enzymes have co-factors. Such as: Metal ions such as Fe2+ and Cu+ Coenzymes/ prosthetic groups such as FAD In some cases, RNA exhibits enzymatic activity and catalyses splicing reaction to produce mature mRNA. Catalytic RNA is called ribozyme. (exception to the rule that most enzymes are proteins). Plateau forms when substrate runs out and the other reason is if the enzyme reaction is reversible and reaches equilibrium. Enzyme kinetics is measured at the initial rate of reaction. Draw a tangent and find the gradient. Rate of reaction in an enzyme can be effected by Substrate concentration Temperature pH for any assay, consider: linearity with time ‘Double the enzyme, double the activity’. If these are not met, it can cause interference. Methods to measure enzyme activity. Continuous assay- allows to measure enzyme activity live and in real time as the reaction proceeds. Discontinuous assay- have to stop the reaction at various points to measure the activity of products formed etc. Tends to use spectroscopy, either substrate or product must absorb light or UV at specific wavelength. Can measure production of product or disappearance of substrate. Coupled reactions. It can be possible that neither enzyme substrate nor the product absorb light. It might be possible to couple the reaction to indicator reaction. The Beer-Lambert law For discontinuous assay, have to set up a different reaction for each time point that needs to be measured. Also need a way to stop the reaction at each time point to measure the activity. = subject to extreme of pH or temperature. Need a way to detect and quantify product formed or substrate used up = can also use spectroscopy, can use ions exchange chromatography to sperate charged products/substrates and radioactive labelling. Effects of substrate concentration on enzyme activity. Rate constants enable us to determine the rate of reaction if we know the substrate concentration(s) Michaelis-Menten equation - can be derived using assumptions and rate equations. Km is NOT the half of Vmax, it is the substrate concentration that gives a rate equal to half of Vmax What is Vmax? Maximal rate of reaction (where the curve plateaus). When all the active sites are saturated. What Is Km? Km is a measure of substrate concentration required for effective catalysis to occur. Enzyme with higher Km requires a higher substrate concentration to achieve a given reaction compared to enzyme with lower Km . How to determine Km and Vmax? Estimate using eye. Computer program used using non-linear regression Lineweaver burk plot. Derivation of Michaelis-Menten equation (not needed for the exam) Assumptions: Always using excess of substrate so conc of substrate is always larger than conc of enzyme. Effect of pH and temperature on enzyme activity. Temperature graph Exceptions can be enzymes from thermophilic bacteria e.g. Taq polymerase used in PCR as it needs to be heat resistant. Changes in pH also unfolds the protein as it denatures and inactivates the enzymes. Some enzymes have a ‘bell shaped’ ph. pH optimum is the highest point on the bell shape. Reason for the bell-shaped curve is the ionisation of 2 residues in the active site. Aspartate 52 Glutamate 35 Both are sides chains of amino acids can be ionised. For the optimal function of enzyme, one residue needs to ionised instead of both.