Enzyme Kinetics Course Materials

1. what are enzymes: Proteins that speed up chemical reactions 2. what is an apoenzyme: protein part of enzyme 3. what is a cofactor: non-protein chemical compound or metallic ion required for an enzymes activity as catalyst 4. what is a holoenzyme: apoenzyme + cofactor 5. what are coenzymes: cofactors that are small organic molecules derived from vitamins - tightly bound: prosthetic grps - loosely bound: cosubstrates 6. enzyme function: speed up chemical reactions by lowering activation energy 7. what are the 6 enzymes classficiations: 1. oxidoreductases 2. transferases 3.hydrolases 4. isomerases 5. lyses 6. ligases 8. enzymes classification - oxidoreductase: oxidoreductase - oxidise/reduce species - oxidase - dehydrogenase 9. enzyme classification - hydrolases: hydrolytically divide substrate - lipase - amylase - pepsin 10. enzyme classification - transferases: move one group onto substrate - (hexo)kinase - phosphatase 11. enzyme classification - isomerase: change spatial orientation - phosphohexose - fumarase 12. enzyme classification - lyase: removal of atoms without hydrolysis - synthase 13. enzyme classification - ligase: join substances (often by condensation) using ATP - synthetase 14. naming enzymes - trivial: - trivial name: suffix -ase added to name of substrate they bind with 15. naming enzymes - systematic: -systematic: precisely identifies enz. enz reactions split into 6 classifications so enz is given a number 1/4 - each classification grp subdivided into grp with 4 digit no - example of named enz = E.C. 2.4.1.18 16. how do enzymes work: - convert substrate to product - product formation determined as a function of time -> product is formed until there is no net change in conc of substrate and product and reaction equilibrium is reached - enz remains unchanged 17. what is the mechanism of enzymatic catalysis: 1. provide reactive surface & suitable environment 2. bring reactants together and position them correctly so they easily attain transition state 3. weaken bonds in reactants 4. reduce energy required for transition state 18. how do enzymes lower activation energy: - substrate must reach transition state before becoming products - when something is stabilised, its energy state is reduced 19. Enzymes as biological catalysts: - all proteins therefore all chemically similar - easily denatured and labile: easily broken -usually specific - specifically inhibited 20. enzymes as inorganic catalysts: - chemically diverse (metals, salts, acids) - stable to heat - often non-specific - usually not inhibited 21. similarities between enzymes as biological catalysts and as inorganic catalysts: - both are unchanged at end of reaction - both can't alter rate of reaction - both can't alter final equilibrium 22. enzyme specificity - absolute: enzymes only binds to a certain substrate e.g enzymes urease only binds to urea 23. enzyme specificity - stereochemical specificity: - only catalyses reaction with one enantiomer 24. enzyme specificity - functional grp specificity: - enzyme only binds 5o substrate with specific functional grps e.g methyl 25. enzyme specificity - low specificity: - catalyse no. of diff reactions involving structurally diverse substrates - e.g glutathione-S-transferase can catalyse reactions towards a large no of substances 26. enzymes as proteins: - are labile - denaturation: oss of bio acitivity 2/4 - change from optimum temp (37) - due to change from optimum pH (7) - exception: pepsin ( stomach) works at pH 2 as stomach is acidic 27. how is enzymes activity measured: - in units -> amount of enz causing transformation of 1 micromol of substrate per min @ 25 temp 28. what is the specific activity of an enz: - no. of units of enz activity per mg of enz protein 29. What is a katal (kat)?: -amount of enzyme activity that converts one mole of substrate per second 30. What is steady state?: when rate of formation and breakdown of enz-substrate complexes are equal 31. what does the plateau on the MM equation show: equilibrium of substrate and product or when all the substrate is used up 32. what will increasing the enzyme conc do to the reaction: increase the reaction velocity as long as there is enough substrate molecules to bind to 33. what will increasing the substrate conc do to the reaction: increase rate of product formation until it reaches Vmax 34. what is Vmax: - the maximum rate of reaction - all available en saturated with substrate - is approached asymptotically -> curve approaches line as it heads to affinity - units: conc per unit time 35. Vmax equation: Kcat[Et] 36. Michaelis-Menten equation: v = (vmax [S])/(Km + [S]) 37. what is Km: - conc of substrate where velocity is at 1/2 Vmax - units: molar M - gives measure of affinity of enz for its substrate - lower Km = greater affinity - most enz Km = 10^-1 - 10^-7 M - depends on: substrate, pH, temp, ionic strength 38. what is the Kcat: - turnover number: how many substrates an enz can turn into product in a second at its max speed - units: sec^-1 39. Kcat equation: Kcat = Vmax/[Et] 3/4 40. what is the enzymes catalytic efficiency equation: Kcat/Km - can be used to see enz preference for diff substrates 41. what is the problem with m-m curve: - is it asymptotic - Vmax is approached asymptotically so it's impossible to obtain definitive value of Vmax and Km from the normal curve graph - straight line graph needed (y=mx+c) 42. Lineweaver-Burk equation: 1/v = (Km/Vmax)(1/[S]) + 1/Vmax - 1/v = y - km/vmax = m - 1/s = x - 1/vmax = c 43. deriving Lineweaver burke plot from M-M equation: look in notes lol 44. competitive inhibition: - bind to active site on enz - bonding = reversible - if substrate increased in can overcome affect of inhibitor - LWB plot: slope increases so km increases - LWB plot: y intercept stays same so vmax unchanged 45. uncompetitive inhibition: binds to an allosteric site on the enzyme-substrate complex - slope unchanged so km stays same - y intercept increases so vmax is decreased 46. non-competitive inhibition (mixed): - either bind allosteric site on enzyme - either bind to allosteric site on ES complex - slope increase so k increases and intercept increases so vmax decreases 47. allosteric enzymes: catalytic site: active site for substrate binding regulatory site: allosteric site for binding of an allosteric effector (modulator) (activator, inhibitor) - doesn't obey M-M kinetics as it has more than one active site - modulator can stimulate or inhibit activity. kclass: km changes. vclass: vmax changes 48. Homotropic allostery: - change in a single catalytic unit can affect other catalytic units. - binding of identical molecules in a cooperative manner 49. heterotopic allostery: - cooperative binding of some other substances as well as the substrate 50. sigmoidal plot: - allosteric enz activity displayed in this plot. 4/4