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Stickase Substrate Transition state X Product If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, but also induces the formation of transition state Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252 Factors affecting Enzymes • substrate concentration In non-enzymic reactions the increase in velocity is proportional to the substrate concentration. Enzymic reaction is faster but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too. • pH Extreme pH levels will produce denaturation and the substrate molecules will no longer fit in active site. Small changes in ionisation and in the charges of the enzyme and it’s substrate molecules will affect the binding of the substrate with the active site. • temperature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation. (Q10 - temperature coefficient - increase in reaction rate with a 10°C rise in temperature. • inhibitors Enzýmová reakcia pri rôznych koncentráciách substrátu EXTREMOPHILES For most enzymes the optimum temperature is about 30°C, many are a lot lower, e.g. cold water fish will die at 30°C because their enzymes denature. A few bacteria have enzymes that can withstand very high temp. up to 100°C. Most enzymes however are fully denatured at 70°C. t vo = [P] / min Unit = mmole/min Slope tan 0 10 20 30 40 Reaction time (min) Specific Activity Units Activity = Protein (mg) y x y = tan x Juang RH (2004) BCbasics S → P mmole Product [P] Enzyme Activity Unit Significance of Enzyme Kinetics v0 = Vmax × K Obtain Vmax and Km 1st order [S] = Low → High Vmax [S] Km + [S] E3 E2 E1 Proportional to enzyme concentration zero order vo = = k3 [Et] × K [S] = Fixed concentration Juang RH (2004) BCbasics 1 2 3 4 5 6 7 8 S + E ↓ P 80 60 40 20 0 0 2 4 6 8 Substrate (mmole) (in a fixed period of time) Product Increase Substrate Concentration 0 Juang RH (2004) BCbasics An Example for Enzyme Kinetics (Invertase) 1) Use predefined amount of Enzyme →E 2) Add substrate in various concentrations → S (x) 3) Measure Product in fixed Time (P/t)→ vo (y) 4) (x, y) plot get hyperbolic curve, estimate → Vmax 5) When y = 1/2 Vmax calculate x ([S]) → Km Vmax 1 vo 1/2 -1 Km 1 Vmax Double reciprocal 1/S Km Direct plot S Juang RH (2004) BCbasics vo A Real Example for Enzyme Kinetics Data Substrate Product Velocity Double reciprocal [S] Absorbance v (mmole/min) 0.25 0.21 → 0.42 0.50 0.36 → 0.72 1.0 0.40 → 0.80 2.0 0.46 → 0.92 no 1 2 3 4 1/S 4 2 1 0.5 1/v 2.08 1.56 1.35 1.16 1.0 v 0.5 0 0 1 2 [S] 2.0 1.0 1/v 1.0 -3.8 0 -4 -2 0 2 1/[S] 4 Juang RH (2004) BCbasics Double reciprocal Direct plot (1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole (2) Reaction time was 10 min Enzyme Inhibitors - chemicals that reduce the rate of enzymic reactions - usually specific, they work at low concentrations - block the enzyme but they do not usually destroy it Irreversible inhibitors: Combine irreversibly with the functional groups of the amino acids in the active site, e.g. nerve gases and pesticides, containing organophosphorus, combine with serine residues in the acetylcholine esterase. Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. - competitive inhibitor competes with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. - non-competitive inhibitor is not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site, e.g. cyanide combines with the Iron in the enzymes cytochrome oxidase, heavy metals Ag or Hg combine with –SH groups. These can be removed by using a chelating agent such as EDTA. Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases Medicine antibiotics, sulphonamides, sedatives, stimulants Affinity labeling of enzymes Enzyme Inhibition (Mechanism) Equation and Description Cartoon Guide I Competitive I Non-competitive Substrate E S S E I Compete for Inhibitor active site S I I Uncompetitive S E I I Different site E + S← → ES → E + P + I ↓↑ EI E + S← → ES → E + P + + I I ↓↑ ↓↑ EI + S →EIS [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition. S I E + S← → ES → E + P + I ↓↑ EIS [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. Juang RH (2004) BCbasics Enzyme Inhibition (Plots) I Competitive I Non-competitive Direct Plots Vmax vo vo I Double Reciprocal Km Km’ I [S], mM Km = Km’ I Uncompetitive Vmax Vmax Vmax’ Vmax’ [S], mM I Km’ Km [S], mM Vmax unchanged Km increased Vmax decreased Km unchanged Both Vmax & Km decreased 1/vo 1/vo 1/vo Intersect at Y axis 1/Km I I I Two parallel lines 1/ Vmax 1/[S] Intersect at X axis 1/Km 1/ Vmax 1/[S] 1/ Vmax 1/Km 1/[S] Juang RH (2004) BCbasics Enzyme Kinetics kcat /Km vo= Vmax [S] Km + [S] kcat Significance zero order 1st order Observe vo change under various [S], resulted plots yield Vmax and Km Turn over number Vmax k3 [Et] 1 mmole min Specific Activity Activity & E3 E2 E1 Km Affinity with substrate Double reciprocal Bi-substrate reaction also follows M-M equation, but one of the substrate should be saturated when estimate the other Inhibition Maximum velocity Activity Unit unit mg Direct plot Competitive Non-competitive Uncompetitive Juang RH (2004) BCbasics Enzýmová analýza Niektoré enzýmy dôležité pre klinickú diagnostiku