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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?
HMG CoA reductase
HMG CoA + NADPH → mevalonate + NADP
Rate limiting, regulatory enzyme in cholesterol synthesis
Cholesterol and heart disease
Cholesterol as key factorExcessive deposition in blood vessels leads
to heart diseaseCarried 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
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
Stroke12%
Coronary heart disease15%
Other CVD9%
Lung cancer 4%
Breast cancer 4%
Colo-rectal cancer 2%
Other cancer14%
Respiratory disease14%
Injuries & poisoning3%
All other causes22%
Risk factors for heart disease
High fat dietOverweight/obeseHigh blood pressureSmokingLack of exerciseHeredity
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)
Ove
rwei
gh
t/o
bes
ity
(%)
16-24
25-34
35-44
45-54
55-64
65-74
75 & over
lifestyleless more
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
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?
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 powdersDrug targetsTests for disease/test kitsClinical tests for liver damage
Biological washing powders
Contain lipases and proteases to digest fat and protein in stainsWork 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-labelledAnti-human antibody
Colour development afteradding 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
+
Tests for liver function
Doctor requests “liver enzymes”Blood testMeasure total protein, albumin and
several enzymes in serum
Liver enzymes
Alanine aminotransferaseAspartate aminotransferase
Enzymes involved in amino acid metabolism
Liver enzymes in blood sample
Alanine aminotransferaseLeaks in to blood from damaged liver
cells e.g. viral hepatitis, paracetamol overdose, fatty liver (alcohol abuse) etc
Aspartate aminotransferaseAlso raised in liver damage but not as
specific
What do we need to know about enzymes?
How to set up an enzyme assayEffects of inhibitorsHow 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
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 Industrialdrug discovery
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