Biochemistry and Metabolism TThe course will cover: EEnzymes – kinetics and control CControl of metabolism CControl of metabolism is the control of enzyme activity

 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  (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

Office of National Statistics (2005) Scotland General Register Office (2005) Northern Ireland General Register Office (2005)

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/m 2 and over Health Survey for England 2004; Scottish Health Survey 2003: Welsh Health Survey 2003/04

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 20 th 2002 – Could this be the cure for obesity?  July 30 th 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 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   +

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 A 340

Kinetics of enzymes “initial rate”

Effect of statin on HMG CoA reductase activity

Competitive inhibition from FEBS Letts (1976)

Enzyme assays in “real life” Hospital pathology lab Industrial drug discovery