Help
Options
Didn't know it?
click below
click below
Knew it?
click below
click below
Don't Know
Remaining cards (0)
Know
retry
shuffle
restart
0:00
Embed Code - If you would like this activity on your web page, copy the script below and paste it into your web page.
Normal Size Small Size show me how
Normal Size Small Size show me how
Enzymes
Biochem and medical genetics
| Question | Answer |
|---|---|
| 3 things cells need to survive | Specific sets of molecules must interact at the right time for the correct duration Specific sets of molecules must change into other sets of molecules at the right time and rate Conformational dynamics at many time scales are needed |
| What is Gibbs free energy | Change in G = Change in H - (T x Change in S) This is a thermodynamic quantity used to determine if a reaction occurs spontaneously. G = amount of energy used in a reaction. Can be calculated by subtracting the energy of the products from the reactants |
| What does Gibbs free energy tell us | A positive G tells you the reaction is not spontaneous and needs energy A negative value tells you the reaction is spontaneous and releases energy When it is 0 the reaction is in equlibrium |
| What do enzymes do | Catalyse specific reactions by providing a lower energy path with an increased rate. They lower the activation energy (or Gibbs free energy required) whilst change in G stays the same |
| The Michaelis - Menton equation | E + S = ES is K1 ES = E + S is K-1 ES = E + P is K2 or Kcat v = (Kcat[E][S])/(Km+[S]) = (Vmax[S])/(Km+[S]) This shows how enzymes function at a certain substrate concentration |
| What does Km mean | Km is then substrate concentration at which half the enzyme active sites are saturated and rate is half Vmax. Units = mmol/dm^3 Large Km = low affinity as enzyme binds weakly to substrate. Small Km = high affinity as enzyme binds strongly to substrate |
| How to find Km and Vmax | A double reciprocal (Lineweaker- Burk plot) is used. Rate is measured at different substrate concentrations and plotted in the form 1/v = 1/Vmax + (Km/Vmax)(1/[S]) Where Km/Vmax is the gradient and 1/Vmax is the y intercept |
| Definitions of Vmax, Kcat | Vmax is the maximum rate possible with that enzyme amount Kcat is the turnover number of the enzyme (how many S converted to P per unit time) Vmax varied with amount of enzyme, Km and Kcat do not |
| What do we use Km, Vmax and Kcat for | Characterising specificity of an enzyme Specificity = Kcat/Km with a high value meaning better catalysis for that substrate Enzymes can have different Km but same Vmax and vice versa, so this gives an indication of their role in metabolism |
| Enzyme role in metabolism based on Km | Hexokinase IV in the liver has a high Km for glucose whilst isoenzymes 1-3 have wider distributions and low Km values. At levels of glucose in someone fasting these are at maximum velocity, whilst in the liver enzymes can deal with further glucose |
| Induced fit mechanisms | Substrate binding plays a role in reorientating active site groups from inactive to active. A precise orientation of groups is needed for catalysis, binding of the substrate may change the 3D structure of the active site giving AAs the correct orientation |
| Selective fit models | The idea that enzymes have an equilibrium between an active and inactive form, with substrates only binding in the active form. This would reduce rate of binding as the concentration of the active form would be lower than that of the enzyme as a whole |
| Structure of the Serine protease enzymes | Contain a catalytic acid bonded to a catalytic base e.g. aspartic acid bound to histidine. This uses serine to catalyse the hydrolysis of amide bonds in unfolded protein molecules |
| Mechanism of action of the serine proteases | His removes H from Ser, leaving electrons which attack the C on the protein. The electrons from the CO bond move to the oxy-anion hole. CN bond destabilised so NH2R leaves (including original H from Ser) |
| Mechanism of action of the serine proteases | Water enters and His again removes the H so OH can bond to the C. This intermediate rearranges to release ser and return the catalyst to is normal state, releasing the second product. |
| Metal ions in catalysis | Enzymes use metal ions for: Electron transfer, radical based rearrangements and redox based reactions. Carbzypeptidase uses an active site Zn2+ ion to polarise an amine carbonyl, allowing better nucleophilic attack by water. |
| Use of cofactors in enzymes | Organic cofactors are large molecules that can undertake complex chemical reactions in short time scales Redox cofactors flavin and NADH Pyridoxamine (B6 AA metabolism) Pantothenic acid (B5 lipid metabolism) Biotin (B7 carboxylation) Thiamine |
| What is vitamin B12 used for | Methylcobalamin - methylation where methyl group is transferred to/from cobalt. Used in methionine synthesis and generation of THF in DNA synthesis Adenosylcobalamin - A source of carbon based radicals used in methylmalonyl-CoA isomerase |
| How can enzymes be regulated | Covalently via chemical modification e.g. proteolysis or phosphorylation Non-covalently via allosteric mechanisms e.g. kinetic effects on Km and Vmax |
| Covalent modification | Proteins are synthesised in a pro or inactive form, often with an additional sequence of AA. The active form is achieved by proteolytic cleavage of the additional sequence |
| Why use covalent modification | To ensure a protein is only active under the correct conditions with the correct stimuli and in the correct location |
| Modification of enzymes in the GI tract | Proteolytic enzymes such as trypsin and chymotrypsin and pepsin are synthesised as zymogens. Proteolysis of these produce changes that expose the active site Trypsin converts chymotrypsinogen to chymotrypsin, which converts trypsinogen to trypsin |
| How is chymotrypsin activated | Chymotrypsinogen is g=cleaved in 3 places Between Lle 16 and Arg 15, with opens that active site by forming new H bonds with Asp Between Tyr 160 and Thr 147 as well as between Ala 149 and Asp 148 to relax the enzyme so it can bind to a substrate |
| Activation of blood clotting factors | Factor X > Xa and prothrombin > thrombin. This is a cascade system which amplifies a response. Factor X is synthesised in the liver using Vit K which cleaves prothrombin in 2 places to form thrombin. Xa binds to cofactor V with Vit K to finish |
| Modification of fibrinogen | Fibrinogen is converted to fibrin following proteolytic cleavage by thrombin and activation of factor XIII. Fibrin has 2 domains which form protofibrils. Factor XIII activated and crosslinks fibrin to form a stable mesh |
| Activation by phosphorylation | AAs with OH groups can accept addition of a phosphate from ATP via protein kinases. This is rapid and reversible Proteins may have many sometimes cooperative sites for phosphorylation. This is important in regulating cell growth, so is a drug target |
| Mechanism of activation via phosphorylation | The AA is unchanged. The transfer of phosphate to OH is energetically favourable, with phosphorylation giving a bulky and negatively charged compound. This phosphorylation causes large conformational changes due to electrostatics which can alter activity |
| Phosphorylation Cascades | Phosphorylation is often associated with intracellular signalling events that result in rapid amplification of signals Cell cycle Glycogen breakdown Transcriptional regulation Cell growth |
| Mechanism of glycogen breakdown | Stimulated by adrenaline, inhibited by insulin. Adrenaline binds to a receptor activating cAMP which starts a phosphorylation cascade converting inactive protein kinases into their active form with each acting on the next. In the end a response is seen |
| Growth factor kinase cascades | Epidermal growth factor binds to its receptor. This causes two chains of tyrosine to move together and be phosphorylated. These must be close together in order for this to happen, which along with the number of steps involved tightly regulated cell growth |
| Allosteric control | The most common form of non-covalent control - when small molecules bind to an enzyme to inhibit/activate them. These can promote/inhibit substrate binding or promote/inhibit release of products |
| Competitive inhibition | A molecule competing for the same binding site as the substrate. This can be overcome by increased substrate concentration. This causes an increase in Km, and has no effect on Vmax |
| Drugs as competitive inhibitors | Drugs tend to mimic the natural enzyme so may reduce activity via competitive binding. Zanamivir - neuraminidase inhibitor Indinavir - HIV protease inhibitor Mevanolin - HMG-CoA inhibitor |
| Key example of a drug as a competitive inhibitor | Imatinib - Tyrosine Kinase inhibitor used to treat chronic myelogenous leukaemia which is caused by a mutation that leads to fusion of the bcr regulator protein with abl kinase, leading to constitutively active bcr-able kinase |
| Allosteric Inhibition | The inhibitor binds to a different site on the protein. This cannot be overcome by increasing substrate concentration. This can occur at a long distance away from the active site. Km unaffected. Vmax reduced |
| Effects of Allosteric ligands | Positive effectors give rise to activation decreasing Km and increasing Vmax Negative effectors give rise to inhibition increasing Km and decreasing Vmax |
| Cooperativity | Allosteric proteins tend to be cooperative They contain multiple identical subunits called oligomers, with long distance interactions between subunits causing a sigmoidal dose response curve. i.e. small change in [S] lead to large changes in enzymes |
| Cooperativities role in sensitivity | The enzyme will reach saturation, but % saturation transitions over a narrower range of [S]. Binding of the first ligand increases the chance of binding a second etc. Homotropic - binding same molecule Heterotopic - binding different molecules |
| Mechanism of cooperativity - MWC model | Assumes subunits of a protein are symmetrically related and each protomer can exist in two forms, a T and R form in equilibrium. Binding of a ligand shifts equilibrium between T and R with the conformational change changing affinity. |
| How allostery works - Phosphofructokinase | Composed of 4 identical subunits. The ligand fructose 6 phosphate binds in each subunit at the interfacial regions. The ATP binding site is near to allow transfer of Pi. Binding of the effectors ATP (-ve) and ADP (+ve) shift equilibrium between T and R |
| Effect of temperature on enzymes | Activity increases to an optimum then falls away. This is due to instability in the protein e.g thermal denaturation due to weak noncovalent forces breaking Body temp is tightly regulated, so this is not a major factor in vivo, but are an issue in fevers |
| Effect of pH on enzymes | Enzymes show a bell shaped curve in response to pH. Cellular pH is regulated so is not an issue. Lysozyme has a pH of 5, where the active site has protonated Glu35 but deprotonated Asp52. This is needed for catalysis but is not need in normal cellular pH |
Created by:
12345678987654321000000
Popular Medical sets