Carbohydrates
A carbohydrate is an organic compound that is composed of atoms of carbon, hydrogen and oxygen in a ratio of 1 carbon atom, 2 hydrogen atoms, and 1 oxygen atom. Some carbohydrates are relatively small molecules, the most important to us is glucose which has 6 carbon atoms. These simple sugars are called monosaccharides.

The primary function of carbohydrates is for short-term energy storage (sugars are for Energy). A secondary function is intermediate-term energy storage (as in starch for plants and glycogen for animals). Other carbohydrates are involved as structural components in cells, such as cellulose which is found in the cell walls of plants.

Two common Monosaccharides, (single sugars) Glucose and Fructose

Hooking two monosaccharides together forms a more complex sugar, such as the union of glucose and fructose to give sucrose, or common table sugar. Compounds such as sucrose are called Disaccharides (two sugars). Both monosaccharides and disaccharides are soluble in water.

Larger, more complex carbohydrates are formed by linking shorter units together to form long or very long sugar chains called Polysaccharides. Because of their size, these are often times not soluble in water. Many biologically important compounds such as starches and cellulose are Polysaccharides. Starches are used by plants, and glycogen by animals, to store energy in their numerous carbon-hydrogen bonds, while cellulose is an important compound that adds strength and stiffness to a plant's cell wall.

Sugars are most often found in the form of a "RING". The glucose molecule in the image above and the one in the image below (Glc) are really the same molecule, just arranged differently. The corners of the "stop sign" represent Carbon atoms even thought they are not labeled with a "C" (its chemistry shorthand). To form these rings, the Carbonyl (C=0) Carbon of the straight-chain form (above) forms a bond with the next to last Carbon in the chain, making the ring.

The image on the left shows two monosaccharides, Glucose and Galactose (Gal). Examine their structure and you will notice there is very little difference. Their molecular formulas, C6H1206, are even the same. Molecules with the same chemical formula, but different molecular structures are called Isomers.

The sugar subunits can be linked by the reaction, dehydration synthesis, to form larger molecules. The disaccharide, Sucrose, is formed from two monosaccharides, Glucose and Fructose.

The disaccharide Lactose is a dimer (two subunits) of Glucose and Galactose, the disaccharide Maltose is a dimer of Glucose.

Large polymers of sugars are called Carbohydrates. Carbohydrates can be 100's of sugars long and either straight or branched. The term Complex Carbohydrate, or sometimes even just Carbohydrate refers to long chains of sugars. Three common types of complex carbo's we will examine are: Starch, Cellulose, and Glycogen. All three are composed only of Glucose. They differ only in the bonding arrangements between the Glucose subunits. Not all complex carbs are composed of glucose alone, many have highly unusual sugars in their chains.

Starch is a long (100's) polymer of Glucose molecules, where all the sugars are oriented in the same direction. Starch is one of the primary sources of calories for humans.

Cellulose is a long (100's) polymer of Glucose molecules. However the orientation of the sugars is a little different. In Cellulose, every other sugar molecule is "upside-down". This small difference in structure makes a big difference in the way we use this molecule.

Glycogen is another Glucose polymer. Glycogen is a stored energy source, found in the Liver and muscles of Humans. Glycogen is different from both Starch and Cellulose in that the Glucose chain is branched or "forked".

 

Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process.

They achieve their effect by temporarily binding to the substrate and, in doing so, lowering the activation energy needed to convert it to a product. The rate at which an enzyme works is influenced by several factors, e.g.,

• the concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in unit of molarity.
• the temperature. As the temperature rises, molecular motion - and hence collisions between enzyme and substrate - speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective.
• the presence of inhibitors.
  • competitive inhibitors are molecules that bind to the same site as the substrate - preventing the substrate from binding as they do so - but are not changed by the enzyme.
  • noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power.
• pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.

The study of the rate at which an enzyme works is called enzyme kinetics. Let us examine enzyme kinetics as a function of the concentration of substrate available to the enzyme.

• We set up a series of tubes containing graded concentrations of substrate, [S].
• At time zero, we add a fixed amount of the enzyme preparation.
• Over the next few minutes, we measure the concentration of product formed. If the product absorbs light, we can easily do this in a spectrophotometer.
• Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of V_i.

Plotting V_i as a function of [S], we find that

• At low values of [S], the initial velocity,V_i, rises almost linearly with increasing [S].
• But as [S] increases, the gains in V_i level off (forming a rectangular hyperbola).
• The asymptote represents the maximum velocity of the reaction, designated V_max
• The substrate concentration that produces a V_i that is one-half of V_max is designated the Michaelis-Menten constant, K_m(named after the scientists who developed the study of enzyme kinetics).

K_m is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the K_m, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

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Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine V_max and K_m.

• V_max is determined by the point where the line crosses the 1/V_i = 0 axis (so the [S] is infinite).
• Note that the magnitude represented by the data points in this plot decrease from lower left to upper right.
• K_m equals V_max times the slope of line. This is easily determined from the intercept on the X axis.

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The Effects of Enzyme Inhibitors

Enzymes can be inhibited

• competitively, when the substrate and inhibitor compete for binding to the same active site or
• noncompetitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency.

The distinction can be determined by plotting enzyme activity with and without the inhibitor present.

Competitive Inhibition

In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while V_max can still be reached if sufficient substrate is available, one-half V_max requires a higher [S] than before and thus K_m is larger.

Noncompetitive Inhibition

With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so

• enzyme rate (velocity) is reduced for all values of [S], including
• V_max and one-half V_max but
• K_m remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.

This Lineweaver-Burk plot displays these results.

An Example

When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme o-diphenol oxidase catalyzes the oxidation of phenols in the apple to dark-colored products. (A similar enzyme, tyrosinase, converts tyrosine to melanin.) Let us determine: 

• the maximum rate at which the enzyme can perform (V_max) and
• the Michaelis-Menten constant (K_m) for this enzyme
  1. when it acts alone. We shall use catechol as the substrate. The enzyme converts it into o-quinone (A), which is then further oxidized to dark products.
  2. when it acts in the presence of a competitive inhibitor. We shall use para-hydroxybenzoic acid (PHBA) (B), which binds the same site as catechol but is not acted upon.
  3. when it acts in the presence of a noncompetitive inhibitor. We shall use phenylthiourea which binds to a copper atom in the enzyme which is essential for its activity.

Preparing for the Assay:

• Grind up pieces of apple and centrifuge the resulting soup.
• The supernatant is your enzyme preparation.
• Because of the speed with which colored products are formed, we can use the intensity of the color as a measure of product formation.
• We measure this in a spectrophotometer, an instrument that measures the absorbance of monochromatic light passed through the sample. Because the products are yellow-brown, they absorb green light (540 nm) best.

First Experiment: No Inhibitor

• Set up four tubes with different concentrations of catechol (the substrate). (Catechol is a relative of the active ingredients in the poison ivy plant and, like them, can cause serious contact sensitivity if it gets on one's skin.)
  • A = 4.8 mM; B = 1.2 mM; C = 0.6 mM; D = 0.3 mM
• Add a fixed amount of enzyme preparation to Tube A and measure the change in absorbance (Optical Density) at 540 nm) at 1 minute intervals for several minutes.
• Record the average change in OD₅₄₀ per minute (Δ OD₅₄₀).
• Because the OD is directly proportional to the concentration of the products, we can use it as a measure of the rate or velocity of the reaction (V_i).
• Repeat with the other three tubes.

	Tube A	Tube B	Tube C	Tube D
[S]	4.8 mM	1.2 mM	0.6 mM	0.3 mM
1/[S]	0.21	0.83	1.67	3.33
Δ OD540 (Vi)	0.081	0.048	0.035	0.020
1/Vi	12.3	20.8	31.7	50.0

The table above summarizes the results. 

Making a Lineweaver-Burk plot of these results shows (red) that

• 1/V_max = 10, so V_max = 0.10
• −1/K_m = − 0.8, so K_m = 1.25 mM
• (In other words, when [S] is 1.25 mM, 1/V_i = 20, and V_i = 0.05 or one-half of V_max.)

Second Experiment: Effect of para-hydroxybenzoic acid (PHBA)

As before, but this time add a fixed amount of a solution of PHBA to each of the four tubes.

The table below summarizes the results.

	Tube A	Tube B	Tube C	Tube D
[S]	4.8 mM	1.2 mM	0.6 mM	0.3 mM
1/[S]	0.21	0.83	1.67	3.33
ΔOD540 (Vi)	0.060	0.032	0.019	0.011
1/Vi	16.7	31.3	52.6	90.9

The Lineweaver-Burk plot of these results is shown above in green.

• 1/V_max = 10, so V_max remains 0.10.
• Now, however, −1/K_m = − 0.4, so K_m = 2.50 mM
• (In other words, it now takes a substrate concentration [S] of 2.50 mM, to achieve one-half of V_max.)

Third Experiment: Effect of phenylthiourea

As before, but this time add a fixed amount of a solution of phenylthiourea in each of the four tubes.

The table below summarizes the results.

	Tube A	Tube B	Tube C	Tube D
[S]	4.8 mM	1.2 mM	0.6 mM	0.3 mM
1/[S]	0.21	0.83	1.67	3.33
ΔOD540 (Vi)	0.040	0.024	0.016	0.010
1/Vi	25	41	62	102

The Lineweaver-Burk plot of these results is shown above in blue.

• Now 1/V_max = 20, so V_max = 0.05.
• But −1/K_m = − 0.8, so K_m = 1.25 mM as it was in the first experiment.
• So once again it only takes a substrate concentration,[S], of 1.25 mM to achieve one-half of V_max.

Summary

Here, then, is a method by which catalytic power of different enzymes can be compared.

The table gives K_m values (mM) for several enzymes - some of which you can encounter with links to other pages on this site.

Enzyme	Substrate	Km (mM)
Catalase	H2O2	1,100
Chymotrypsin	Gly-Tyr-Gly	108
Carbonic anhydrase	CO2	12
beta-galactosidase	D-lactose	4
Acetylcholinesterase	acetylcholine (ACh)	0.09
beta-lactamase	benzylpenicillin	0.02

 

The favoured model for the enzyme–substrate interaction is the induced fit model.This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding. These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lower the energy of the reaction's transition state, by providing an alternative chemical pathway for the reaction. Mechanisms of catalysis include catalysis by bond strain: by proximity and orientation: by active-site proton donors or acceptors: covalent catalysis and quantum tunnelling.

Enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. However, some kinetic data can suggest possibilities to be examined by other techniques. For example, a ping–pong mechanism with burst-phase pre-steady-state kinetics would suggest covalent catalysis might be important in this enzyme's mechanism. Alternatively, the observation of a strong pH effect on V_max but not K_m might indicate that a residue in the active site needs to be in a particular ionisation state for catalysis to occur.

An important goal of measuring enzyme kinetics is to determine the chemical mechanism of an enzyme reaction, i.e., the sequence of chemical steps that transform substrate into product. The kinetic approaches discussed above will show at what rates intermediates are formed and inter-converted, but they cannot identify exactly what these intermediates are.

Kinetic measurements taken under various solution conditions or on slightly modified enzymes or substrates often shed light on this chemical mechanism, as they reveal the rate-determining step or intermediates in the reaction. For example, the breaking of a covalent bond to a hydrogen atom is a common rate-determining step. Which of the possible hydrogen transfers is rate determining can be shown by measuring the kinetic effects of substituting each hydrogen by deuterium, its stable isotope. The rate will change when the critical hydrogen is replaced, due to a primary kinetic isotope effect, which occurs because bonds to deuterium are harder to break then bonds to hydrogen.^[25] It is also possible to measure similar effects with other isotope substitutions, such as ¹³C/¹²C and ¹⁸O/¹⁶O, but these effects are more subtle.

Isotopes can also be used to reveal the fate of various parts of the substrate molecules in the final products. For example, it is sometimes difficult to discern the origin of an oxygen atom in the final product; since it may have come from water or from part of the substrate. This may be determined by systematically substituting oxygen's stable isotope ¹⁸O into the various molecules that participate in the reaction and checking for the isotope in the product. The chemical mechanism can also be elucidated by examining the kinetics and isotope effects under different pH conditions,^[26] by altering the metal ions or other bound cofactors, by site-directed mutagenesis of conserved amino acid residues, or by studying the behaviour of the enzyme in the presence of analogues of the substrate(s).