Protein Function: Enzymes

What are enzymes?
Do enzymes have to be proteins?

• most enzymes are proteins although some nucleic acids (ribozymes) have catalytic properties.
• the first enzyme crystallized was urease by Sumner in 1922. He showed it was a protein. I guess its just serendipidy that the first organic chemical synthesized was urea!
  What will be the reaction catalyzed by urease?
• more recently Thomas Cech and coworkers showed that some RNA molecules, such as the small nuclear RNA's (snRNA) can self-splice (see Nucleic Acid notes) and behave just like an proteinaceous enzyme: such RNA's are called ribozymes.

• enzymes are extremely efficient biological catalysts

• catalysts accelerate rates of reactions without being changed by the reaction.

• Enzymes accelerate rates of reaction without being changed by the reaction.
• Enzymes accelerate rates of reaction without changing the equilibrium position of the reaction.
• Typically enzymes carry out a chemical reaction, such as the transformation of substrates (reactants) to products, A-->B, through an enzyme-substrate complex.
• Rates are accelerated 10^3 to 10^17-times compared to the uncatalyzed rate.

What are the properties of enzymes as catalysts?

• enzymes accelerate rates of reactions anywhere from 1x10^3 to 1x10^17-fold depending on the enzyme.
• enzymes do not alter the equilibrium position for a reaction.
• enzymes are highly specific for their substrates: they are stereospecific for binding of reactant and stereoselective for the formation of the correct stereoisomer of the product.
• there are few side-reactions.
• yields approach 100%.
• the individual reaction catalyzed is a small, discrete step.
  • For example, the enzyme urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide: and the enzyme acetylcholine esterase, which is important in the propagation of a nerve impulse across the motor-neural junction, catalyzes the hydrolysis of the ester acetyl choline into acetic acid and choline. What is an ester?
  • The sum of a sequence of enzymes, each carrying out a small step, is a large chemical change. This is metabolic pathway: the sum of all the small enzyme catalyzed steps in a metabolic pathway produces a large chemical transformation. For example, the complete combustion of glucose to CO2 and H2O requires 19 different enzymes and is completed in 33 steps (some of which are duplicated).

What is a substrate?

• The substrate is the reactant in an enzyme catalyzed reaction. A ligand is a general term for any substance that binds to a protein. Substrates are ligands that bind to proteins and undergo a specific chemical reaction.

  For example, if E is the enzyme, we may describe this chemical transformation of A to B as:
  E + A --> EA -->TS --> EB --> E + B
  where EA and EB are enzyme-substrate complexes and TS is an anzyme-bound transition state for the chemical transformation of A to B.

How do enzymes accelerate rates?

• Hydrolysis of a protein to amino acids occurs rapidly during digestion (order of minutes) yet protein in solution is stable to hydrolysis at 37C even under acid conditions (order of days). How long does Jello last in your fridge compared to your stomach?
• Similarly sucrose is stable in your pantry and even in solution, yet its hydrolysis to glucose and fructose is energeticallly favorable.
• Enzymes lower the energy barrier for a reaction, the energy of activation.
• Enzymes lower the energy of activation of a reaction which accelerates rates by decreasing the time taken to reach equilibrium or (net) completion of the reaction.

Where on the enzyme does the chemical transformation of the substrate occur?

• The substrate binds to the enzyme in a very specific manner at the active site.

• the active site is a cleft or pocket in the 3-D structure of a protein, often between subunits or domains.

What is the function of the 3-D structure of a protein?

• It brings specific amino acid residues together in 3-D space, to the same 3-D location, in such a way that they have a complementary fit to the substrate.
• Specific reactive groups (functional groups) which are the side-chains of the amino acid residues of the enzyme come together in close proximity and the correct orientation to bind the substrate.
• These specific functional groups catalyze the chemical transformation of the substrate.

• non-covalent bonds that form between the substrate and enzyme. Covalent bonding does occur in some cases.
• there is a complementary fit between the shape of the active site and the shape of substrate. Stereospecificity between the shape of the active site and the shape of substrate.
• this allows formation of specific non-covalent bonds between active site and substrate.
• a good analogy is the specificity between a lock and key, although because the 3-D structure of proteins and enzymes is flexible, the binding of substrate to enzyme can and does induce a conformational change in the protein and the transition state in the substrate

How do we measure enzyme catalyzed reactions?

• Constant, fixed amount of enzyme
• Vary concentration of substrate (reactant) so that we can measure how the rate varies with the concentration of reactant. This will enable us to determine the kinetic constants for the enzyme.
• Measure loss of reactant (substrate) or appearance of product
• We are measuring rates, rate at which reactant disappears or rate at which product appears -- accurate timing of events: units will be moles/unit time
• Graphical presentation of data: substrate concentration will be the independent variable -- we set the concentrations -- plot on the x axis, and rate of reaction (velocity, v) will be the dependent variable -- what we measure -- plot on the y axis

What are the main features of enzyme kinetics?

• remember Oreo lyase!
• if the amount of an enzyme is kept constant, variation in the concentration of substrate correlates with the rate of formation of the product.
• the graph of velocity, v, versus substrate concentration, [S], is a rectangular hyperbola.
• Saturation kinetics: the rate approaches an upper limit, the maximum velocity, because the enzyme becomes saturated with substrate.

• E+S<-->ES-->E+P
• The rate of reaction, v, is a function of the concentration of enzyme-substrate complex, [ES]
• Maximum rate, Vmax, occurs when the enzyme is saturated with substrate, that is when all of the enzyme is present in the ES complex
• The basic equation describing enzyme kinetics was derived by Michaelis and Menten and was based on an assumption of a steady state for the concentration of the ES complex proposed by Briggs and Haldane: this Michaelis-Menten equation is
  • v = Vmax.[S]/(Km + [S])
• where Vmax is the maximum velocity (the upper limiting value of v) and Km is a constant, the Michaelis constant.
• Km is the substrate concentration at half maximum velocity,
  for when v = Vmax/2,
  the equation v = Vmax.[S]/(Km + [S]) collapses to Km = S
• Enzymes have Km values that approximate substrate concentration in the cell, that is Km = [S] in vivo.
• Why is that?
  • it allows the response to changes in [S] to be approximately linear
• it is difficult to derive numerical values for the constants Vmax and Km from a curved graph. It is easy to derive them from a straight line graph. We can change the rectangular hyperbola graph of the Michaelis-Menten equation into a straight line graph of the form y=mx+b, where m = the slope and b = intercept on the y axis, by taking reciprocals:
  • 1/v = {Km/(VmS)} + (S/VmS) = {(Km/Vm)1/S} + 1/Vm
  • and a plot of 1/v vs 1/S yields a straight line of slope Km/Vm and intercept 1/Vm. This double reciprocal plot is called the Lineweaver-Burk plot.

How do drugs inhibit enzyme catalysed reactions?

• Pharmaceutical industry, rational drug design through understanding enzyme mechanisms.
• for the Oreo lyase experiment, the chocolate chip cookie decreased the rate of reaction with the correct substrate because it looks like an ooreo but cannot undergo the lyase (splitting) reaction.

• Competitive. Competition between S and I for binding to enzyme decreases rate of reaction, v. Overcome by increasing [S] which means that the parameter Km is increased. The effect of a chocolate chip or or Fig Newtons on oreo lyase! Diagnostically, a decrease in Km and no change in Vm.
• Non-competitive. The inhibitor binds both to free E and to ES complex and decreases the rate of the reaction, v. Not overcome by increasing [S] which means that the parameter Vm is decreased. Diagnostically, a decrease in Vm and no change in Km.
• Irreversible: poisons like nerve gas that inhibits acetylcholine esterase.

What is site-directed mutagenesis?

• Change the base sequence in the gene so that you exchange one amino acid for another. That is you carry out a mutation of the gene, but it is very specific -- you have chosen which position in the gene and which amino acid you are going to change -- hence site-directed mutagenesis. If that amino acid is at the active site of the enzyme you may alter the kinetics or the specificity or the mechanism of the enzyme catalyzed reaction.
• A tool used to understand how enzymes work.

What are the six classes of enzymes?
Enzyme class is based on reaction type.
Enzyme names end in -ase usually as *reaction type*-ase. For example, hydrolysis = hydrolase

1. oxidation and reduction: oxidoreductases, commonly the addition or removal of H

• Lactate --> pyruvate + 2[H]
• ethanol -->acetaldehyde + 2[H] (see chemistry notes)

2. transfer of a chemical group: transferases, eg kinases

• formation of a phosphate ester
• R-OH + ATP --> R-O-PO4 + ADP, phosphoryl group transfer to an alcohol.

3. hydrolysis: hydrolases

• dipeptide + H2O --> 2[amino acids]
• sucrose --> glucose + fructose

4. two reactions of opposite type, eliminations and additions

• splitting or elimination: lyases
  • decarboxylations, removal of carboxyl group
  • pyruvate --> acetaldehyde + CO2

• joining or addition: synthase
  • ADP + phosphate = ATP

5. interconversion of isomers: isomerases

• L-amino acid --> D-amino acid.

6. addition reactions requiring energy: synthetases

• energy in the form of ATP is required for the synthesis the amide functional group of glutamine, that is for the synthesis of the amino acid glutamine from the amino acid glutamate: no part of ATP is incorporated into the product glutamine -- it simply serves to provide energy to drive the reaction
• L-glutamate + NH3 + ATP --> L-glutamine + ADP + phosphate

Enzyme kinetic calculation

The kinetics of an enzyme catalyzed reaction are determined in the presence and absence of an inhibitor at different substrate concentrations. You obtain the following data:

	v (millimoles/min)
[S](millimoles)	control	inhibitor
1.25	1.72	0.98
1.67	2.04	1.17
2.50	2.63	1.47
5.00	3.33	1.96
10.00	4.12	2.38

Estimate Vmax and Km in the presence and absence of the inhibitor.
What kind of inhibition is involved (competitive or non-competitive)?



Plot the data as v vs [S], velocity versus substrate concentration.

From the Michaelis-Menten plot (upper panel), inhibition is non-competitive, the decrease in Vmax even at high substrate concentrations being diagnostic for this type of inhibition. (Note: competitive inhibition is overcome by high substrate concentrations)



Numerical estimates of Vmax and Km are made from a linear plot, the double reciprocal or Lineweaver-Burk plot (lower panel).

The intercept on 1/v is 0.197 for the control (circles) so that Vmax = 5.1 mmole/min.
Note: you cannot get a good estimate of Vmax from the Michaelis-Menten plot.
For the control data the slope (Km/Vmax) is 0.481 and Km = 2.4 mM (Vmax x slope)

For the data with inhibitor, the intercept on 1/v is 0.337 and Vmax = 3.0 mmole/min.
The slope is 0.858 and Km = 2.5 mM
A decrease in Vmax with the same Km is diagnostic of non-competitive inhibition

From the intercept on 1/S axis, -1/Km is -0.4 and Km is 2.5 mM

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Bich 107 lecture notes on Enzymes were last updated 10/06/03

Comments to Martyn Gunn