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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) “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