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
Enzyme Kinetics
Biochemistry, Medicine, Phase 1
| Term | Definition |
|---|---|
| Enzyme kinetics | study of the rate of an enzyme catalysed reaction and how it varies |
| Rate of catalysed reaction depends on | (1) different substrate concentrations (2) amounts of inhibitors (3) metal ions and cofactors (4) pH |
| Reaction rate (V) | Δ[product]/[Δtime] or [Δsubstrate/[Δtime] |
| ΔG | activation energy |
| Rate 1 (k1) | K[ES][S] |
| Rate 2 (k2) | Rate [ES] |
| At low substrate concentration | the reaction is directly proportional to the substrate concentration (1st order kinetics) |
| At high substrate concentrion | the reaction is independent of the substrate concentration (zero order kinetics); the enzymes will be saturated and wont be able to react more quickly |
| V0 | initial reaction velocity, measured as soon as a substrate is mixed; rate of formation of the product |
| Vmax | maximal velocity of an enzyme catalysed reaction, ie when all the enzyme active sites are fully saturated with substrate |
| Km | the rate they come together to form the enzyme substrate; Michaelis kostant; the lower the Km the better the enzyme is at working when substrate concentrations are small; (unit miliMolar) |
| Km = | (k-1 + k2)/k1 |
| [S] | substrate concentration |
| V0 = | (Vmax[S])/(Km+[S]) |
| If V0 = 0.5Vmax (1) | 0.5 Vmax = (Vmax[S])/(Km+[S]) |
| If V0 = 0.5Vmax (2) | Km |
| Steady state assumption | [ES] is constant; formation of ES = Loss of ES |
| Formation of ES + | R1 + R-2 |
| Loss of ES = | R-1 + R2 |
| R1 | Very small, negligible, meaning ES > E + P Rather than ES <> E + P |
| If V0=Vmax | then [E]total = [ES] |
| Michaelis-Menton equation | Vo=(Vmax[S])/(Km+[S]) |
| Where Vo = 1/2Vmax | Km is the [S] |
| Catalytic efficiency; Specificity Constant | Kcat/Km |
| Kcat | enzyme turnover number; how many substrates and enzyme can turn into product at its max speed; measured in /sec; based on a single enzyme molecule; catalase high number (40 million) |
| Kcat = | Vmax / Etotal |
| Assumption 1 | [S] >> [E] so that the amount of substrate bound by enzyme at any one time is small |
| Assumption 2 | [ES] does not change with time (steady-state), formation of ES = breakdown of ES (to E+S and E+P) |
| Assumption 3 | initial velocity used, concentration of product small and back reaction of P to S can be ignored |
| Substrate affinity | When k2 is rate limiting k2<<k-1, the Km equation reduces to k-1/k1, which is defined as the dissociation constant (kd), of the ES complex; then, Km does represent a measure of affinity of the enzyme for its substrate in the complex |
| kd | dissociation constant; when k2<<k-1; normally k2=k-1 or k2>>k-1; normally km is not Km a simple measure of substrate affinity; normally Kcat is required |
| High Km | enzyme is not doing a lot of work; substrate highly efficient on its own; the activation energy of the reaction is low, e.g. catalase for H202 and chymotrypsin and glycyltryosinylglycine; hexokinase is much higher |
| Comparing catalytic effciency | neither Km or Kcat alone are sufficient to compare catalytic efficiency of two enzymes; two enzymes may have the same Kcat but very different Km; catalytic must be measured by specificity constant |
| Lineweaver-Burke plot | rearranging the Michaelis-Menton equation to the form y=mx+c; 1/Vo = (Km/Vmax[S]) + 1(Vmax); adds graphical value to the constant; can be used to determine the Km and Vmax as well to determine the action of enzyme inhibitors |
| x-axis of Lineweaver-Burke plot | 1/[S] |
| Intercept of Lineweaver-Burke plot on y-axis | 1/Vmax |
| Intercept of Lineweaver-Burke plot on x-axis | -1/Km |
| y-axis of Lineweaver-Burke plot | 1/Vo |
| Clinical uses of enzyme measurements | differential diagnosis of disease by investigating plasma levels of 'escaped' enzymes ; laboratory estimations of metabolites such as glucose in body fluids (blood, urine); e.g. glucose oxidase is used in Diastix (plasma) and Clinistix (urine) tests |
| Lactate dehydrogenase | clinical uses in differential diagnosis; composed of four monomers; each monomer can either be heart or muscle type; five different isozymes of lactate hydrogenase exist: H4, H3M, H2M2, HM3 and M4 |
| Appearance of creatine kinase and lactate dehydrogenase in plasma after myocardial infarction | increase in CK2 activity 24 hours after; increase in LDH activity 48 hours after |
| Isozymes of lactate dehydrogenase | can be separated by chromatography; different tissues have different levels of each of the isozyme |
| Isozymes of lactate dehydrogenase and myocardial infarction | During, endothelial cells rupture, releasing their contents into the blood stream; this increases the serum levels for lactate hydrogenase isozyme 1 and 2 and cane be used as a diagnostic tool |
| Enzyme inhibitors | competitive inhibitors block the enzyme active |
| Malonate | inhibits succinate dehydrogenase; competitive inhibitor; takes up the active site, blocking succinate (the substrate) from entering the succinate dehydrogenase |
| Non-competitive (reversible or irreversible) enzyme inhibitors | interfere in some other way with the catalytic mechanisms; does not directly block the active site; irreversible poisons the enzyme |
| Addition of a chealator (EDTA) | reversible non-competitive enzyme inhibitor; inhibits Mg2+ requiring enzyme |
| Organophosphorus | inhibits cholinesterase irreversibly; poisons the enzyme |
| Competitive inhibitors | alter the apparent Km not the Vmax; increase the [S] it takes to reach Vmax; reduces the steepness of the slope |
| Non-competitive inhibitors | you cannot reach the same Vmax but Km and Vmax/2 is the same |
| Clinical uses of enzyme inhibitors | control of angiotensin production (cleavage of Angiotensin I to Angiotensin II results in peripheral vasoconstriction and aldosterone secretion); treatment of heart failure with ACE inhibitors prevents sodium/water retention > oedema |
| Enzyme inhibitors as toxins | mustard gas, sarin (nerve gases) |
| Regulation of enzyme activity | allosteric binding sides (+ or - effectors), pharmacological altering of enzyme activity; covalent modification by other enzymes (phosphorylation - kinases or dephosphorylation - phosphates); induction or repression of enzyme synthesis |
| Allosteric regulation | one of the most common forms of regulation; good way of controlling enzyme activity; sigmoid curve (compared to hyperbolic curve of M-M kinetics); slow to start, but more rapid change that reaches Vmax quicker |
| Allosteric activator | opens up active site, making it more open to substrate; can be positive or negative (more or less effective active site) |
| Negative allosteric effectors eg | ATP and citrate of phosphofructokinase (metabolism) |
| Positive allosteric effectors eg | phosphoenolpyruvate (PEP) and fructose 1,6 bis phosphate on pyruvate kinase |
| Allosteric regulation of PFK and PK | key elements of glycolysis; the conversion of fructose 1,6-phosphate to fructose 1,6 bis phosphate REQUIRES ATP (co-substrate and allosteric regulator), so final product includes ADP; using PFK; |
| AMP | can act as positive regulator during glycolysis if there is not enough ATP; if you have a lot of ATP and citrate, you do need to put more carbon changes (aka fructose 1,6-phosphate) through glycolysis because you have enough energy |
| Fructose 1,6-bis phosphate | also acts as a positive allosteric regulator of phosphenyolpyruvate to pyruvate (a later step in glycolysis); good example of co-ordinated regulation |
| Covalent modification | most common form is the addition or removal of phosphate from Ser, Thr, Tyr, His residues |
| Phosphorylation (regulation of glucose metabolism); single cascade system | examples of covalent modification; increases activity of glycogen phosphorylase, which is responsible for degrading glycogen; it occurs by phosphorylase kinase serine 14 and requires ATP; ALSO reduces activity of glycogen synthase (synthesises glycogen) |
| Dephosphorylation of serine 14 | by phosphorylase phosphatase |
| Glycogen synthase kinase 3 | phosphorylates at several serine resides; inactivates the enzyme entirely |
| Glucose metabolism | coordinated regulation of synthesis and breakdown of glycogen via adrenaline and glucagon; shows how protein phosphorylation may be increasing or decreasing activity of even catalysis of a different reaction |
| Comment covalent modifcations | Adenylylation or AMPylation (Tyr residues, ATP to PPi); Uridylylation (Tyr residues, UTP to PPi) |
| PPi | pyrophosphate |
| Residue | monomer |
| Regulation of amount of enzyme (example) | high blood glucose levels lead to an increase in insulin production; insulin increases rate of synthesis of key enzymes involved in glucose metabolism = glucokinase, phosphofructokinase, pyruvate kinase |
| Effect of substate availability; speed | Change in velocity; immediate |
| Effect of product inhibition; speed | Change in Vm and/or Km; immediate |
| Effect of allosteric control (usually end product) | Change in Vm and/or Km; immediate |
| Effect of covalent modification (another enzyme) | Change in Vm and/or Km; immediate to minutes |
| Effect of synthesis or degradation (hormone or metabolite) | Change in amount of enzyme; hours to days |
Created by:
emmaallde
Popular Biochemistry sets