TBL 2 Enzyme Kinetics Student Guide 2020 PDF

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Summary

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Description

Molecules, Cells and Systems

4PY019

Enzyme Kinetics Student Study Guide

Facilitator

References

Dr B. Wright

Required This study guide

Contact Details

Lecture Slides on 4PY019 MCS CANVAS course

[email protected]

Video Clips An Introduction to Enzyme Kinetics https://www.youtube.com/watch?v=X_YXTWU2maY Steady States and the Michaelis Menten Equation https://www.youtube.com/watch?v=7u2MkbsE_dw Enzymatic Inhibition and Lineweaver Burke Plots https://www.youtube.com/watch?v=ALU24yhKJZw

Learning Outcomes

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Molecules, Cells and Systems

4PY019

This learning pack considers enzymes and how they catalyse reactions that are crucial for cell function in addition to their role as potential targets for drug action. On completion of this topic, you should be able to:

Recognise a variety of cell locations where enzymes operate

Understand the concept of the active site and the enzyme-substrate complex

Recognise the biochemical features of enzymes essential to catalysis

Understand the effect of pH on the side chain groups of amino acids and how this affects enzyme function

Recognise the Michaelis-Menten Equation and linear transformations

Understand the concept of Km

Understand the concept of Vmax

Understand the units of Km and Vmax

Use the Lineweaver-Burke plot to estimate Km and Vmax

Recognise the different modes of enzyme inhibition and their effects on Km and Vmax

Team-Based Learning

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Molecules, Cells and Systems

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Content 1. Active sites, amino acid side chains and enzyme optima 2. The Michaelis-Menten equation 3. Physiological significance of Km 4. Linear transformations of the Michaelis-Menten equation 5. Enzyme inhibitors 6. Effect of inhibitors on Km and Vmax

1.

Active sites and amino acid side chain structures

Enzymes are proteins. Amino acids are the building blocks of proteins. At the “active site” of the enzyme (the place where catalysis takes place) are amino acid side chains. The ones that are most likely to be present are those which are capable of interacting with positive and negative charges and with water. Many enzyme reactions use water as a means to break bonds - hydrolysis - or produce it during catalysis – condensation. Similarly the environmental conditions that the enzyme finds itself in will also affect its catalytic efficiency. The ionic concentration and prevailing pH will both dramatically affect enzyme activity with optimum conditions existing for all enzymes. In addition to the amino acid side chains, active sites will nearly always contain a divalent ion such as zinc, manganese, cobalt or selenium. These are held in place by co-ordinate bonds often formed with histidine residues.

2.

The Michaelis-Menten equation

v = Vmax . [S] / Km + [S]

Arises from a rectangular hyperbola Thus

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Molecules, Cells and Systems

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Which can be described by the general equation:

Vmax v

=

Km

+ 1

where Vmax and Km are constants

S

The curve is known as a Michaelis-Menten plot and the equation describing it as the MichaelisMenten equation. The constant K m is known as the Michaelis constant. It has a value that is characteristic for each enzyme with a particular substrate, and has the units of substrate concentration, i.e. molarity. For most cellular enzymes these values are measured in millimoles, micromoles or less. The Michaelis constant is an approximate measure of the affinity of the enzyme for the substrate. The relationship is reciprocal, so that a large Michaelis constant indicates low affinity for substrate and a small Michaelis constant a high degree of affinity. In general more accurate results can be derived from plotting straight-line graphs than from curves and for this reason, linear derivations of the Michaelis-Menten equation have been obtained.

Study the Lecture Power Point slides on CANVAS: “Introduction to Enzymes” Watch the Video Clip on “An Introduction to Enzyme Kinetics” You spend 2 hours on this activity.

3.

Physiological significance of Km

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Molecules, Cells and Systems

4PY019

Glucose is a very important fuel for all cells. The normal resting physiological concentration of glucose in the blood is about 5 mM – that’s about 90 mg per 100 mL. After a meal this can rise to anything between 15 and 25 mM – depending on how many Mars  bars you eat. Now the liver is able to take up much of this glucose and store it as glycogen, maybe as much as 500 g. This is achieved via the enzyme GLUCOKINASE which has a Km value for glucose of about 10 mM. Glucose reserves in the muscles and liver will only last about a day, so when glucose is scarce, the brain gets priority on it – as it is virtually dependent on this sugar as an energy source. This is due to brain cells possessing the enzyme HEXOKINASE, which has a Km value of approximately 0.1 mM for glucose. Thus the brain can scavenge what little glucose is around in times of actual starvation or metabolic starvation as seen in diabetes.

4.

Linear transformations of the Michaelis-Menten equation

Lineweaver-Burke

1 v

=

Km S.Vmax

+

1 Vmax

5

Molecules, Cells and Systems Hanes-Woolfe

4PY019 S v

Eadie-Hofstee

=

S

+

Vmax

v = Vmax

Km Vmax

-

Km.v S

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4PY019

The Hanes-Woolfe plot, with S on the x-axis and S/v on the y-axis, is considered the best to use for estimating kinetic parameters (why might this be?) while the Eadie-Hofstee plot is similar to the Scatchard plot that is often used in pharmacology to estimate receptor numbers through quantification of ligand (often hormone) receptor binding (Bmax analogous to Vmax) and the affinity of a the ligand for the receptor the Kd (also measured in units of concentration).

Study the Lecture Power Point slides on CANVAS : “The Michaelis Menten Equation” Watch the Video Clip: “Steady States and the Michaelis Menten Equation” You should spend 2 hours on this activity

5.

Enzyme Inhibition

In pharmacy, many enzymes are the targets of drugs. The active site is an obvious location for an interaction. If this is the case the drug may – but not necessarily - act as a competitive inhibitor, often because it has a chemical resemblance to the substrate. However drugs may also operate through interactions with separate sites close to or distant from the active site. These are called allosteric sites. These type of drugs are usually – though not exclusively – non-competitive inhibitors. They often have chemical structures that are dissimilar to the substrate.

6.

Effect of inhibitors

The effect of a competitive inhibitor on the Km of an enzyme is to increase it. However this mode on inhibition may be overcome by increasing the substrate concentration sufficiently high that the Vmax value may be reached – i.e. there is no effect on Vmax by a competitive inhibitor. For a noncompetitive inhibitor there is no effect on the enzyme’s Km, but the Vmax is lowered.

Study the Lecture Power Point slides on CANVAS: “Enzyme Kinetics” Watch the Video Clip on “Enzymatic Inhibition and Lineweaver Burke Plots” You should spend 2 hours on this activity.

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