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What Characteristic Features Define Enzymes?

• Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality • Enzymes are the agents of metabolic function • Catalytic power, specificity, regulation


Reaction profile showing large ∆G‡ for glucose oxidation, free energy change of -2,870 kJ/mol; catalysts lower ∆G‡, thereby accelerating rate.

The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis. Enzyme 4, fructose 1,6, biphosphate aldolase, catalyzes the C-C bond- breaking reaction in this pathway.


A 90% yield over 10 steps, for example, in a metabolic pathway, gives an overall yield of 35%. Therefore, yields in biological reactions must be substantially greater; otherwise, unwanted by-products would accumulate to unacceptable levels.

Enzyme Classification


• organic compounds essential in the diet in small amounts • have little or no caloric value • chemical composition is varied • normally classified according to their polarity


Classification of Vitamins
• Fat-soluble vitamins (nonpolar) Vitamin A Vitamin D Vitamin E Vitamin K • Water-soluble vitamins (polar) Vitamin C Vitamin B Complex

Fat-Soluble vitamins: A, D, E, K
• • • • Soluble in fatty tissues Stored in the body for long periods of time Not easily excreted Can be overconsumed (overdose)


Water-Soluble Vitamins: C and B Complex • Soluble in water • Excreted in the urine and pose little threat of overdose • However, they must be consumed in sufficient amounts on a daily basis

Nutritional Minerals
• elements,other than C, H, N, and O, needed for good health. • many are present as ions rather than as neutral atoms • Major minerals (~4% of the body’s weight) Ca, P, Mg, Na, K, Cl, and S • Minor minerals Fe, Cu, Zn, I, Se, Mn, F, Cr, and Mo


Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way?

• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! • Urease is a good example: – Catalyzed rate: 3x104/sec – Uncatalyzed rate: 3x10 -10/sec – Ratio is 1x1014 !

• Enzymes selectively recognize proper substrates over other molecules • Enzymes produce products in very high yields - often much greater than 95% • Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield


The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has slightly alkaline pH optimum, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimum near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with pK' near 6 is essential to its activity. Might it be a histidine residue within the active site?

The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a function of temperature. The decrease in the activity above 50° is due to C thermal denaturation.


How Can Enzymes Be So Specific?
• “Lock and key” was the first explanation for specificity • “Induced fit” provides a more accurate description • Induced fit favors formation of the transitionstate intermediate


Several terms to remember
• • • • • rate or velocity rate constant rate law order of a reaction molecularity of a reaction

The Transition State
Understand the difference between ∆G and ∆G‡
• The overall free energy change for a reaction is related to the equilibrium constant • The free energy of activation for a reaction is related to the rate constant • It is extremely important to appreciate this distinction!


Energy diagram for a chemical reaction (A→P) and the effects of (a) raising the temperature from T1 to T2 or (b) adding a catalyst. Raising the temperature raises the average energy of A molecules, which increases the population of A molecules having energies equal to the activation energy for the reaction, thereby increasing the reaction rate. In contrast, the average free energy of A molecules remains the same in uncatalyzed versus catalyzed reactions (conducted at the same temperature). The effect of the catalyst is to lower the free energy of activation for the reaction.

What Equations Define the Kinetics of Enzyme-Catalyzed Reactions?

• Enzymes accelerate reactions by lowering the free energy of activation • Enzymes do this by binding the transition state of the reaction better than the substrate


The Michaelis-Menten Equation
• Louis Michaelis and Maud Menten's theory • It assumes the formation of an enzymesubstrate complex • It assumes that the ES complex is in rapid equilibrium with free enzyme • Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S


Understanding Km
• • • • The "kinetic activator constant" Km is a constant Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding


Understanding Vmax
• • • • The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased

The dual nature of the Michaelis-Menten equation
Combination of 0-order and 1st-order kinetics • When S is low, the equation for rate is 1st order in S • When S is high, the equation for rate is 0order in S • The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!


The turnover number
A measure of catalytic activity • kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. • If the M-M model fits, k2 = kcat = Vmax/Et • Values of kcat range from less than 1/sec to many millions per sec


The catalytic efficiency
Name for kcat/Km An estimate of "how perfect" the enzyme is • kcat/Km is an apparent second-order rate constant • It measures how the enzyme performs when S is low • The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together

Enzyme Units
• IU (International Unit) – amount of enzyme that catalyze the formation of 1 micromole of product in 1 minute • Katal – amount of enzyme that converts 1 mole of substrate to product in 1 second • Specific activity – enzyme unit per mg of protein


Linear Plots of the Michaelis-Menten Equation

• • • • •

Lineweaver-Burk Eadie-Hofstee Hanes-Woolf Hanes-Woolf is best - why? Smaller and more consistent errors across the plot

The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope.


A Hanes-Woolf plot of [S]/v versus [S], another straight-line rearrangement of the MichalelisMenten equation.

Eadie Hofstee Linear Plot


What Can Be Learned from the Inhibition of Enzyme Activity?
• Enzymes may be inhibited reversibly or irreversibly • Reversible inhibitors may bind at the active site or at some other site • Enzymes may also be inhibited in an irreversible manner – Penicillin is an irreversible suicide inhibitor

Lineweaver-Burk plot of competitive inhibition, showing lines for no I, [I], and 2[I]. Note that when [S] is infinitely large (1/[S] = 0), Vmax is the same, whether I is present of not. In the presence of I, the negative
x-intercept = -1 [I]   Km  1 +  KI  


Structures of succinate, the substrate of succinate dehydrogenase (SDH), and malonate, the competitive inhibitor. Fumarate (the product of SDH action on succinate) is also shown.

Lineweaver-Burk plot of pure noncompetitive inhibition. Note that I does not alter Km but that it decreases Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).


Lineweaver-Burk plot of pure uncompetitive inhibition. Note that I decreases Km and Vmax. In the presence of I, the yintercept is equal to (1/Vmax)(1 + I/KI).

Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidase, which catalyzes an essential step in bacterial cell wall synthesis. Penicillin consists of a thiazolidine ring fused to a β-lactam ring to which a variable group R is attached. A reactive peptide bond in the β-lactam ring covalently attaches to a serine residue in the active site of the glycopeptide transpeptidase. (The conformation of penicillin around its reactive peptide bond resembles the transition state of the normal glycoprotein peptidase substrate.) The penicilloyl-enzyme complex is catalytically inactive. The bond between the enzyme and penicillin is indefinitely stable; that is, penicillin binding is irreversible.


Are All Enzymes Proteins?
Relatively new discoveries • Ribozymes - segments of RNA that display enzyme activity in the absence of protein – Examples: RNase P and peptidyl transferase • Abzymes - antibodies raised to bind the transition state of a reaction of interest