Post on 07-May-2015
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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
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?
from Drug Discovery Today 11 481-493 (2006)
HMG CoA reductase
HMG CoA + NADPH → mevalonate + NADP
Rate limiting, regulatory enzyme
in cholesterol synthesis
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)
Benefits of Lipitor
Lowers total cholesterol by ~ 40%.
Lowers "bad," low-density lipoprotein (LDL) cholesterol by ~ 50%.
Reduces risk for heart attack and stroke
Every five minutes somebody in the UK dies from a heart attack
Office of National Statistics (2005)
Scotland General Register Office (2005)
Northern Ireland General Register Office (2005) www.heartstats.org
Deaths by cause, women, 2004, United Kingdom
Stroke
12%
Coronary heart disease
15%
Other CVD
9%
Lung cancer
4%
Breast cancer 4%
Colo-rectal cancer 2%
Other cancer
14%
Respiratory disease
14%
Injuries & poisoning
3%
All other causes
22%
Risk factors for heart disease
High fat diet
Overweight/obese
High blood pressure
Smoking
Lack of exercise
Heredity
Increase in obesity
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
Prevalence of overweight and obesity by age, women, latest
available year, England, Scotland and Wales
0
10
20
30
40
50
60
70
80
England (2004) Scotland (2003) Wales (2003/04)
Overw
eig
ht/
ob
esit
y (
%)
16-24
25-34
35-44
45-54
55-64
65-74
75 & over
lifestyle less more
diet
Food Standards Agency
traffic light labelling
Guideline daily amount
Metabolic syndrome
1. 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
Metabolic syndrome
Prevalence – 20% of adults in US
Risk Factors: poor eating habits,
lack of exercise, genetics, ageing
Metabolic syndrome
1. Increased risk of heart disease
2. Increased risk of Type 2 diabetes
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?
Complex interplay between glucose/fat/cholesterol
metabolism
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)
Biochemistry and Metabolism
Why should we study enzymes?
Why are enzymes interesting?
How do enzymes feature in daily life?
Enzymes?
Biological washing powders
Drug targets
Tests for disease/test kits
Clinical tests for liver damage
Biological washing powders
Contain lipases and proteases to digest fat and protein in stains
Work at lower temperatures
Drug targets
Inhibitors of enzyme – COX-2 (cyclooxygenase-2,
responsible for prostaglandin synthesis)
ELISA
Enzyme Linked ImmunoSorbant Assays
Used widely for detection of proteins and antibodies in patient samples
Uses enzyme conjugated to antibody
Test for HIV
Patients with HIV have antibodies against the viral proteins
Detect these antibodies using ELISA
ELISA for antibodies in HIV
HIV proteins
Patient sample –
Containing anti HIV
Enzyme-labelled
Anti-human antibody
Colour development after
adding substrate for enzyme
ELISA
Detection antibody has enzyme attached to it
e.g. alkaline phosphatase – substrate 4-nitrophenyl phosphate
NO2
O
P O
OH
OHO
NO2
O
P O
OH
OH
colourless yellow
+
96 well plate for ELISA
Pregnancy test kits
Tests for liver function
Doctor requests “liver enzymes”
Blood test
Measure total protein, albumin and several enzymes in serum
Liver enzymes
Alanine aminotransferase
Aspartate aminotransferase
Enzymes involved in amino acid metabolism
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
What do we need to know about enzymes?
How to set up an enzyme assay
Effects of inhibitors
How to interpret the results
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
Kinetics of enzymes
0.0 2.5 5.0 7.5 10.0 12.50
1
2
3
4
5
6
time
NA
DP
H (
A3
40)
―initial rate‖
Effect of statin on HMG CoA reductase activity
0.000 0.025 0.050 0.075 0.1000
5
10
15
20
25
control
+ statin
[substrate]
rate
Effect of statin on HMG CoA reductase activity
Competitive inhibition
from FEBS Letts 72 323-326 (1976)
Enzyme assays in “real life”
Hospital pathology lab Industrial
drug discovery