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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 |