Enzymes

Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part.)

• Catalase. It catalyzes the decomposition of hydrogen peroxide into water and oxygen.

  H₂O₂ -> H₂O + O₂

  A single molecule of catalase can break 5.6 million molecules of hydrogen peroxide each minute.
  
  
• Carbonic anhydrase. It is found in red blood cells where it catalyzes the reaction

  CO₂ + H₂O <-> H₂CO₃

  It enables red blood cells to transport carbon dioxide from the tissues to the lungs. [Discussion]

  A single molecule of carbonic anhydrase can process 36 million molecules of substrate each minute.

• hydrogen bonds
• ionic interactions
• and hydrophobic interactions

Most of these interactions are weak and especially so if the atoms involved are farther than about one angstrom from each other. So successful binding of enzyme and substrate requires that the two molecules be able to approach each other closely over a fairly broad surface. Thus the analogy that a substrate molecule binds its enzyme like a key in a lock.

This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.

Competitive inhibition

The necessity for a close, if brief, fit between enzyme and substrate explains the phenomenon of competitive inhibition.

Enzyme cofactors

• Some of these are metal ions such as Zn²⁺ (the cofactor for carbonic anhydrase), Cu²⁺, Mn²⁺, K⁺, and Na⁺.
• Some cofactors are small organic molecules called coenzymes. The B vitamins
  • thiamine (B1)
  • riboflavin (B2) and
  • nicotinamide
  are precursors of coenzymes.

Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its catalytic act.

Lysozyme: a model of enzyme action

• N-acetylglucosamine (NAG)
• N-acetylmuramic acid (NAM)

Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft.

• With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.
• Some hydrogen bonds also form with the C=O groups of several peptide bonds.
• In addition, hydrophobic interactions may help hold the substrate in position.

X-ray crystallography has shown that as lysozyme and its substrate unite, each is slightly deformed. The fourth hexose in the chain (ring #4) becomes twisted out of its normal position. This imposes a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings 4 and 5. It is just at this point that the polysaccharide is broken. A molecule of water is inserted between these two hexoses, which breaks the chain. Here, then, is a structural view of what it means to lower activation energy. The energy needed to break this covalent bond is lower now that the atoms connected by the bond have been distorted from their normal position.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the "lock" as well as the "key" changes shape as the two are brought together. (This is sometimes called "induced fit".)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H⁺ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.

Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H⁺ and OH^- ions. [Discussion] The hydrogen ion (H⁺) left over can replace that lost by Glu-35.

The reaction is now complete. The chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location of the bacterial cell wall and continue its work of digesting it.

Factors Affecting Enzyme Action

• tertiary structure (i.e. shape) in enzyme function and
• noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

• the protease pepsin works best as a pH of 1-2 (found in the stomach) while
• the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents). [Discussion]

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO^- of Asp-52, the catalytic action of lysozyme would cease.

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Regulation of Enzyme Activity

Anchoring enzymes in membranes

• the plasma membrane
• the membranes of mitochondria and chloroplasts
• the endoplasmic reticulum
• the nuclear envelope

Inactive precursors

Feedback Inhibition

Precursor Activation

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

• In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.
• In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.

Regulation of Enzyme Synthesis

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on - or off - the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase - by transcribing and translating the necessary gene(s) - and so can begin to metabolize the sugar.