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 Today11 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 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

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

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

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)

Ove

rwei

gh

t/o

bes

ity

(%)

16-24

25-34

35-44

45-54

55-64

65-74

75 & over

lifestyleless 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 disease2. 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 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

96 well plate for 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

+

96 well plate for ELISA

Pregnancy test kits

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

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 Industrialdrug discovery