Transcript Aims of lecture

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)
“statins”
Inhibitors of the enzyme HMG CoA reductase
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
Deaths by cause, women, 2004, United Kingdom
Injuries & poisoning
3%
Respiratory disease
14%
All other causes
22%
Other cancer
14%
Colo-rectal cancer 2%
Breast cancer 4%
Lung cancer
4%
Coronary heart disease
15%
Other CVD
9%
Office of National Statistics (2005)
Scotland General Register Office (2005)
Northern Ireland General Register Office (2005)
Stroke
12%
www.heartstats.org
Risk factors for heart disease
High fat diet
Overweight/obese
High blood pressure
Smoking
Lack of exercise
Heredity
Increase in obesity
Prevalence of overweight and obesity by age, women, latest
available year, England, Scotland and Wales
16-24
80
25-34
35-44
45-54
70
55-64
65-74
Overweight/obesity (%)
60
75 & over
50
40
30
20
10
0
England (2004)
Scotland (2003)
Wales (2003/04)
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
lifestyle
less
more
diet
Guideline daily amount
Food Standards Agency
traffic light labelling
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
96 well plate for ELISA
ELISA for antibodies in HIV
Enzyme-labelled
Anti-human antibody
Colour development after
adding substrate for enzyme
Patient sample –
Containing anti HIV
HIV proteins
ELISA
Detection antibody has enzyme
attached to it
e.g. alkaline phosphatase – substrate
4-nitrophenyl phosphate
NO2
NO2
O
+
O
HO P O
OH
O
OH
colourless
HO P O
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
6
NADPH (A340)
5
“initial rate”
4
3
2
1
0
0.0
2.5
5.0
7.5
time
10.0
12.5
Effect of statin on HMG CoA
reductase activity
25
control
rate
20
+ statin
15
10
5
0
0.000
0.025
0.050
[substrate]
0.075
0.100
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