Biochemistry and Metabolism

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Biochemistry and Metabolism

• The course will cover
• Enzymes kinetics and control
• Control of metabolism
• Control of metabolism is the control of enzyme
  activity

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Biochemistry and Metabolism

• Why should we study metabolism?
• Why is metabolism interesting?
• Where does metabolism feature in daily life?
• Why should we study enzymes?
• Why are enzymes interesting?
• How do enzymes feature in daily life?

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from Drug Discovery Today 11 481-493 (2006)
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statins Inhibitors of the enzyme HMG CoA
reductase
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HMG CoA reductase
HMG CoA NADPH ? mevalonate NADP Rate
limiting, regulatory enzyme in cholesterol
synthesis
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Cholesterol and heart disease

• Cholesterol as key factor
• Excessive deposition in blood vessels leads to
  heart disease
• Carried as lipoproteins
• bad cholesterol low density lipoprotein (LDL)
• good cholesterol high density lipoprotein
  (HDL)

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Benefits of Lipitor

• Lowers total cholesterol by 40.
• Lowers "bad," low-density lipoprotein (LDL)
  cholesterol by 50.
• Reduces risk for heart attack and stroke

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Every five minutes somebody in the UK dies from a
heart attack
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Office of National Statistics (2005) Scotland
General Register Office (2005) Northern Ireland
General Register Office (2005)

www.heartstats.org
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Risk factors for heart disease

• High fat diet
• Overweight/obese
• High blood pressure
• Smoking
• Lack of exercise
• Heredity

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Increase in obesity
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Overweight/obesity BMI 25 kg/m2 and over Health
Survey for England 2004 Scottish Health Survey
2003 Welsh Health Survey 2003/04
www.heartstats.org
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(No Transcript)
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lifestyle
more
less
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diet
Guideline daily amount
Food Standards Agency traffic light labelling
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Metabolic syndrome

• Obesity, particularly around the waist (having an
  "apple shape")
• 2. Elevated blood pressure
• 3. An elevated level of triglycerides and a low
  level
• of high-density lipoprotein (HDL) the "good"
  cholesterol
• 4. Resistance to insulin

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Metabolic syndrome
Prevalence 20 of adults in US
Risk Factors poor eating habits, lack of
exercise, genetics, ageing
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Metabolic syndrome

• Increased risk of heart disease
• Increased risk of Type 2 diabetes

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Questions to ask about diet and metabolism

• How does a high carbohydrate diet lead to
  obesity?
• How does a high fat diet lead to heart disease?
• What is disturbed in diabetes?

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Complex interplay between glucose/fat/cholesterol
metabolism
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Need to understand diet and metabolism

• Daily Mail
• August 20th 2002 Could this be the cure for
  obesity?
• July 30th 2003 Chemical could burn away obesity
• Gillian McKeith she hasn't a clue about
  nutrition (Brit Dietetic Assn)

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Biochemistry and Metabolism

• Why should we study enzymes?
• Why are enzymes interesting?
• How do enzymes feature in daily life?

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Enzymes?

• Biological washing powders
• Drug targets
• Tests for disease/test kits
• Clinical tests for liver damage

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Biological washing powders
Contain lipases and proteases to digest fat and
protein in stains Work at lower temperatures
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Drug targets
Inhibitors of enzyme COX-2 (cyclooxygenase-2,
responsible for prostaglandin synthesis)
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ELISA

• Enzyme Linked ImmunoSorbant Assays
• Used widely for detection of proteins and
  antibodies in patient samples
• Uses enzyme conjugated to antibody

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Test for HIV

• Patients with HIV have antibodies against the
  viral proteins
• Detect these antibodies using ELISA

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96 well plate for ELISA
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ELISA for antibodies in HIV
Enzyme-labelled Anti-human antibody
Colour development after adding substrate for
enzyme
?
?
Patient sample Containing anti HIV
HIV proteins
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ELISA

• Detection antibody has enzyme attached to it
• e.g. alkaline phosphatase substrate
  4-nitrophenyl phosphate

yellow
colourless
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96 well plate for ELISA
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Pregnancy test kits
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Tests for liver function

• Doctor requests liver enzymes
• Blood test
• Measure total protein, albumin and several
  enzymes in serum

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Liver enzymes

• Alanine aminotransferase
• Aspartate aminotransferase
• Enzymes involved in amino acid metabolism

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Liver enzymes in blood sample

• Alanine aminotransferase
• Leaks in to blood from damaged liver cells e.g.
  viral hepatitis, paracetamol overdose, fatty
  liver (alcohol abuse) etc
• Aspartate aminotransferase
• Also raised in liver damage but not as specific

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What do we need to know about enzymes?

• How to set up an enzyme assay
• Effects of inhibitors
• How to interpret the results

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HMG CoA reductase assay
HMG CoA NADPH ? mevalonate NADP Assay
from loss of absorbance at 340 nm as NADPH
consumed Mix enzyme and substrate in cuvette and
read A340
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Kinetics of enzymes
initial rate
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Effect of statin on HMG CoA reductase activity
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Effect of statin on HMG CoA reductase activity
Competitive inhibition
from FEBS Letts 72 323-326 (1976)
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Enzyme assays in real life
Hospital pathology lab
Industrial drug discovery