Enzymes Kinetics and Inhibition Enzymes How do we characterize an enzyme? Biological catalyst
Highly specific Mostly proteins Enzyme Classification Common name Not always informative about reaction catalyzed Ex. Chymotrypsin
Systematic name Enzyme Commission 6 major classes Ex. Transferases Classification number EC 2.7.3.2 2=transferase, 7=phosphate, 3=nitrogen group as acceptor, 2=unique
Enzyme Classification Enzymes What are enzyme cofactors? Non-protein component Organic group or metal When is a cofactor a prosthetic group? Holoenzyme versus apoenzyme
Enzymes How do metal ion cofactors function? Bridging group, coordination complex Stabilize conformation in active form
Vitamins are parts of coenzymes Thiamine pyrophosphate Nicotinamide adenine dinucleotide Pyridoxal phosphate Enzymes and Free Energy What does G tell us about the nature of a reaction?
What does it mean if G is = 0? What is true if G is positive? Calculating Free Energy A+B C+D
G = G0 + RT ln[C][D] / [A][B] ln G0 = standard free energy change; A, B, C, D present at 1M G0/ = at pH 7 Calculating Free Energy At equilibrium 0 = G0/+ RT ln[C][D] / [A]
ln [B] Since Keq = [C][D]/ [A][B] We can get G0/ = -2.303RT log10Keq Calculating Free Energy Enzymes
Do not change equilibrium of reactions, speed up both forward and reverse reactions Equilibrium depends upon free energy difference between products and reactants Enzymes
How do enzymes speed up reactions? Enzymes decrease activation energy Enzyme Kinetics What evidence
supports the existence of an enzymesubstrate complex? Enzyme Kinetics Images from x-ray crystallography Enzyme Kinetics
Change in spectroscopic characteristics Enzyme Kinetics What do we know about the active site of an enzyme?
where substrate binds three-dimensional cleft small part of enzyme substrate attached by weak bonds Enzyme Kinetics What does the lock and key model represent?
Enzyme Kinetics How is it different from the induced fit model? Michaelis Menten Equation Enzyme E combines with substrate S to form enzyme substrate complex ES and ES breaks
down to form E and product P E + S k1 k-1 ES k2
P+E Michaelis-Menten Equation Initial velocity equals rate of breakdown of ES V0 = k2 [ES] Must define ES in other terms Formation of ES = k1[E][S]
Breakdown of ES = (k-1 + k2 )[ES] At steady state: k1[E][S] = (k-1 + k2 )[ES] Michaelis-Menten Equation Rearrange equation [E][S]/[ES] = (k-1 + k2 )/k1
Define new term Km Michaelis-Menten constant Km = (k-1 + k2 )/k1 we now substitute in above and solve for [ES] [ES] =[E][S]/Km [E] = [E]T [ES] Michaelis-Menten Equation Substitute for[E]
[ES] =([E]T [ES])[S]/Km Solve for [ES] [ES] = [E]T [S]/[S] + Km Going back to previous equation v 0=k2[ES] V0 = k2 [E]T [S]/[S] + Km Maximal rate Vmax is when [ES] =[E]T V0 = Vmax [S]/[S] + Km
Michaelis-Menten Equation V0 = Vmax [S]/[S] + Km When initial velocity = maximal velocity Km = [S] Michaelis-Menten Equation
For a particular enzyme, can determine Km by varying substrate concentration keeping enzyme concentration constant and measuring initial velocity of reaction Lineweaver Burk Equation
Use double reciprocal plot Enzyme Kinetics What do we know about Km?
Not a fixed value Varies with structure of substrate Varies with pH, temperature, ionic strength Each substrate has characteristic Km Km usually between 10-1 and 10-6 M Derived from rate constants Enzyme Kinetics E
+ E + S S k 1 k1 k -1k -1 ES
ES k2 k 2 P P If k-1 is considerable larger than k2 so that Km = k-1/k1 this represents the dissociation constant of ES complex
Therefore, Km tells us about strength of ES complex high Km = weak bonding; low Km = strong bonding Enzyme Kinetics What does Vmax represent? Turnover number
What is the kinetic constant or kcat? Kcat = Vmax/[E]0 [E]0 = initial conc. Enzyme # of substrate molecules converted into product in unit time when enzyme is saturated When [S] Km, kcat/Km can be used as measure of catalytic efficiency