6.5 Enzymes

Enzyme Structure and Function

Enzymes are biological catalysts that speed up the chemical reactions cells need to survive. Without them, most metabolic reactions would happen far too slowly to sustain life. Their ability to lower activation energy, bind specific substrates, and be regulated by the cell makes them central to virtually every process in metabolism.

Enzyme Catalysis in Metabolism

Enzymes work by lowering the activation energy of a reaction, which is the minimum energy needed for that reaction to proceed. They don't change whether a reaction happens (that's determined by thermodynamics), just how fast it happens. A few key properties:

• Enzymes are not consumed in the reactions they catalyze. A single enzyme molecule can process thousands of substrate molecules.
• Enzymes are highly specific. Each enzyme typically catalyzes one reaction or one type of reaction, determined by the shape and charge of its active site.
• Enzymes operate within metabolic pathways, which are sequences of enzyme-catalyzed reactions that convert starting molecules into final products. Examples include glycolysis, the citric acid cycle, and the electron transport chain. In these pathways, the product of one enzyme becomes the substrate for the next.

Structure-Function of Enzyme Active Sites

Enzymes are proteins (though some RNA molecules called ribozymes also have catalytic activity). Because they're proteins, their function depends directly on their shape, which comes from their structure at every level:

• Primary structure: the amino acid sequence, which determines how the protein folds
• Secondary structure: local folding patterns like $\alpha$-helices and $\beta$-sheets
• Tertiary structure: the overall 3D shape of the polypeptide, which forms the active site
• Quaternary structure: the arrangement of multiple polypeptide subunits (only in some enzymes)

The active site is a specific region on the enzyme where the substrate binds and the chemical reaction takes place. It's formed by amino acid residues brought together by the enzyme's tertiary structure, even if those residues are far apart in the primary sequence.

Substrate binding at the active site involves several types of non-covalent interactions: hydrogen bonds, electrostatic interactions, van der Waals forces, and hydrophobic interactions. Together, these create both the specificity (only the right substrate fits) and the affinity (the substrate binds tightly enough to be held in place).

Two models describe how enzymes and substrates interact:

• Lock and key model: the active site and substrate have perfectly complementary shapes from the start, like a key fitting into a lock.
• Induced fit model: the active site changes shape slightly when the substrate binds, wrapping around it for a tighter fit. This is the more widely accepted model and helps explain why enzymes can stabilize the transition state of a reaction.

Some enzymes also require non-protein helpers called cofactors (metal ions like $Zn^{2+}$ or $Mg^{2+}$) or coenzymes (organic molecules, often derived from vitamins, like $NAD^+$) to function properly.

Enzyme Regulation and Inhibition

Cells need to turn enzyme activity up or down depending on conditions. If every enzyme ran at full speed all the time, the cell would waste energy and resources. Regulation keeps metabolism balanced and responsive.

Factors Regulating Enzyme Activity

Enzyme concentration: More enzyme molecules means more active sites available, which increases the reaction rate (assuming substrate is available).

Substrate concentration: Adding more substrate increases the reaction rate, but only up to a point. Once every active site is occupied, the enzyme is saturated, and adding more substrate won't speed things up. This maximum rate is called $V_{max}$.

Temperature: Each enzyme has an optimal temperature where it works fastest. Below that, molecular motion is slower and fewer productive collisions occur. Above that, the enzyme begins to denature, meaning its 3D structure unfolds and the active site loses its shape. For most human enzymes, the optimum is around 37°C.

pH: Each enzyme also has an optimal pH. Changes in pH alter the ionization of amino acid side chains in the active site, which can disrupt substrate binding and catalysis. For example, pepsin (a stomach enzyme) works best at pH ~2, while trypsin (in the small intestine) works best at pH ~8.

Enzyme Inhibition

Inhibitors are molecules that reduce enzyme activity. They're important both in cellular regulation and in medicine (many drugs are enzyme inhibitors).

• Competitive inhibitors bind to the active site, directly blocking the substrate. They compete with the substrate, so increasing substrate concentration can overcome competitive inhibition.
• Non-competitive inhibitors bind to a site other than the active site (an allosteric site), changing the enzyme's shape so it can't catalyze the reaction as effectively. Increasing substrate concentration does not overcome this type of inhibition.
• Uncompetitive inhibitors bind only to the enzyme-substrate complex (not the free enzyme), locking the substrate in place and preventing the reaction from completing.

Allosteric Regulation and Other Controls

Allosteric regulation involves molecules binding to a site on the enzyme that is not the active site. This binding changes the enzyme's conformation:

• Allosteric activators shift the enzyme into a more active shape.
• Allosteric inhibitors shift it into a less active shape.

Covalent modifications can also switch enzymes on or off. The most common example is phosphorylation, where a phosphate group is added to the enzyme by a kinase. Removal of the phosphate by a phosphatase reverses the effect. Acetylation is another example.

Feedback inhibition is a common regulatory strategy in metabolic pathways. The end product of the pathway acts as an inhibitor of an enzyme early in the pathway. This prevents the cell from overproducing a product it already has enough of. For instance, in the pathway that synthesizes the amino acid isoleucine, isoleucine itself inhibits the first enzyme in the pathway (threonine deaminase).

Enzyme Kinetics

Enzyme kinetics is the study of how fast enzyme-catalyzed reactions proceed and what factors influence that speed. The reaction rate (also called velocity) depends on:

• Substrate concentration
• Enzyme concentration
• Temperature
• pH
• Presence of inhibitors or activators

At low substrate concentrations, the rate increases steeply as you add more substrate. As substrate concentration rises, the rate levels off and approaches $V_{max}$. The substrate concentration at which the reaction rate is half of $V_{max}$ is called the Michaelis constant ($K_m$). A low $K_m$ means the enzyme reaches half its maximum speed at a low substrate concentration, indicating high affinity for its substrate.