XVII. Biological catalysis: enzymes

Catalysis

faster not more !

Most reactions take place with the help of third compounds, helpers or catalysts.

Catalysts help and only help reactions to proceed, they do not have any effect on reaction equilibrium.

They do not get used up during the reaction.

They increase only the speed, the rate, with which the reaction approaches equilibrium

We’ve seen examples of biological catalysts: ATPases, kinases, polymerases.

Collectively, biological catalysts are called enzymes.

The effect catalysts have on the reaction is to lower its activation energy.

Activation energy is a hump in an energy profile between products and substrates that must be overcome before the reaction proceeds. Activation energy exists for both exergonic and endergonic reactions

Mechanism of Catalysis

Reactions proceed faster because:

the enzyme increases the concentration of substrates by holding two molecules in physical proximity;

correct geometry of the substrates is established on the enzymatic surface;

chemical bonds are strained by interaction with the electron clouds of substrates.

Active Site

opposites attract

· The part of the enzyme where all this happens is referred to as the active site.

· It is a patch of specific residues, geometrically and electrically complementing the substrates.

Geometric complementation is best illustrated by the fatty acid phospholipase, which has a long tunnel in its structure into which fatty acid acyl chain can fit.

Electrical interaction between substrate and enzyme are best seen in ATPases, in which charged residues interact with negatively charged phosphate groups.

Induced fit

fit or make it fit ?

Complementarity between enzyme and substrate is not a rigid one. Most enzymes adapt themselves to fit the substrate better only after binding to it. The fit is more like that of a glove to a hand (induced fit) than that between lock and key.

Enzyme Kinetics

Investigation of the rate of reaction as a function of substrate concentration reveals whether a reaction is an enzymatic one and how the enzyme is interacting with the substrate.

The plot of rate versus concentration shows binding and processing stages of enzymatic reaction.

At low concentrations, when the number of enzyme molecules is higher than that of the substrate, the increase of the rate reflects binding affinity between the two. The more they like each other, the quicker enzyme-substrate complex will be formed, and the quicker the reaction will proceed. A measure of the affinity, Km, is the concentration of the substrate at which the rate proceeds at 50% of it maximum.

At high substrate concentrations, the rate reaches maximum, Vmax. At this stage all enzyme molecules are saturated with the substrate, binding is irrelevant, and the rate is determined by the speed of processing.

Activity Modulators

Enzymatic activity can be affected by things other than substrate.

Temperature

Some like it hot, some like it cold.

Higher temperatures increase probability of collisions (good), weaken bonds in the substrates (good), but also could cook the enzyme (very bad).

pH

Some like it sour, some like it basic.

For example, proteolytic enzymes work best at low pH (< 5); at pH 7 they are completely inactive.

Ligand binding

In addition to binding substrates, enzymes bind other things, ligands.

Competitive inhibitors

impostors

When the ligand binds to the active site and prevents a legitimate substrate from binding, we call it a competitive inhibitor.

feedback inhibition

Noncompetitive inhibitors

When a ligand binds so strongly to an active site that substrate can’t dislodge it, we call it a noncompetitive inhibitor.

Allosteric interactions

Action at a distance

Some ligands bind to places other than active sites (regulatory sites).

Binding of a ligand (e.g. Ca) to a regulatory site changes the shape of the inactive enzyme, either stimulating or inhibiting enzymatic reaction.

cooperativity

Enzymatic Helpers

Proteins often provide a structural framework for a catalytic group, but are not themselves catalysts. The catalysts are either (i) prosthetic groups, (ii) cofactors, or (iii) coenzymes.

Prosthetic groups

non-proteins, strongly held

Prosthetic groups are ions or non-protein catalysts that are covalently attached to a protein.

Cofactors

loose assembly

· Cofactors are more loosely held catalysts.

Coenzymes

missing partners

Some cofactors are non-protein organic molecules (coenzymes), for example:

NAD: nicotinamide adenine dinucleotide, H+ carrier.

FAD: flavin adenine dinucleotide, H+ carrier.

CoA: coenzyme A, acetyl group carrier.

CoQ: ubiquinone, hydrogen carrier.