Metabolism and Biocatalysis

Overview: From Nutrients to Cellular Work

Metabolism is the entirety of chemical reactions in a living system that convert nutrients into energy and building blocks, and dispose of waste. Biocatalysis is the acceleration of these reactions by biological catalysts, mainly enzymes.

In this chapter the focus is:

• How metabolism is organized (catabolism vs anabolism, metabolic pathways)
• How cells capture and use energy (ATP and “high‑energy” compounds)
• How enzymes act as biocatalysts (mechanisms, specificity, regulation)
• Basic examples of metabolic pathways to illustrate principles
  (detailed structures of biomolecules themselves are covered elsewhere)

Organization of Metabolism

Catabolism and Anabolism

Metabolism is commonly divided into two complementary parts:

• Catabolism
• Degradative pathways.
• Convert energy‑rich nutrients (carbohydrates, fats, proteins) into smaller, simpler molecules (e.g. $CO_2$, $H_2O$, $NH_3$).
• Release free energy, often stored temporarily in $ATP$ or other “energy‑carrier” molecules.
• Examples: glycolysis, fatty acid β‑oxidation, citric acid cycle (in its energy‑harvesting role).
• Anabolism
• Biosynthetic pathways.
• Use small precursor molecules (e.g. pyruvate, acetyl‑CoA, amino acids, nucleotides) to build complex cellular components (proteins, nucleic acids, lipids, polysaccharides).
• Consume energy, usually from $ATP$ and reducing equivalents like $NADPH$.
• Examples: protein biosynthesis, fatty acid synthesis, nucleotide synthesis.

Many pathways are amphibolic: they function both catabolically and anabolically depending on cellular needs (e.g. citric acid cycle provides both energy and biosynthetic precursors).

Metabolic Pathways and Networks

Individual reactions are organized into metabolic pathways:

• Each step is catalyzed by a specific enzyme.
• The product of one reaction is the substrate of the next.
• Pathways are often linear, branched, or cyclic (e.g. citric acid cycle).
• Pathways intersect extensively, creating a metabolic network.

Common design features:

• Irreversible steps: Certain reactions have strongly negative Gibbs free energy change ($\Delta G \ll 0$) in cells and act as control points.
• Committed steps: Early, essentially irreversible steps that commit metabolites to a particular pathway.
• Compartmentation: In eukaryotic cells, pathways are separated into organelles (e.g. fatty acid oxidation in mitochondria, fatty acid synthesis in cytosol), allowing independent regulation and avoiding futile cycles.

Energy in Metabolism: ATP and High‑Energy Compounds

Chemical Energy and Free Energy Changes

Biological systems use chemical reactions to perform work (mechanical, osmotic, biosynthetic). The thermodynamic quantity that determines whether a reaction can perform useful work at constant temperature and pressure is the Gibbs free energy change, $\Delta G$.

• Exergonic reactions: $\Delta G < 0$; can proceed spontaneously (thermodynamically favored).
• Endergonic reactions: $\Delta G > 0$; require an input of energy to proceed.

Cells couple exergonic and endergonic reactions so that the overall process has $\Delta G < 0$.

ATP: The Universal Energy Currency

Adenosine triphosphate ($ATP$) is the central energy currency in cells.

• Structure: adenine base + ribose + three phosphate groups.
• Key reaction (ATP hydrolysis):

$$
ATP + H_2O \rightarrow ADP + P_i
$$

or

$$
ATP + H_2O \rightarrow AMP + PP_i
$$

where $P_i$ is inorganic phosphate and $PP_i$ is pyrophosphate.

• Under physiological conditions, ATP hydrolysis has a significantly negative $\Delta G$, making it a powerful “energy donor.”

Important points:

• ATP is not “high‑energy” in an absolute sense; it simply has a phosphoryl group transfer potential that is high relative to many other molecules in cells.
• ATP is continuously recycled: a human body turns over its own weight in ATP each day, even though total ATP content at any instant is much smaller.

Coupling Reactions via ATP

Many biosynthetic reactions are endergonic when considered alone. Cells drive them by coupling to ATP hydrolysis.

Example: phosphorylation of glucose to glucose‑6‑phosphate:

1. Unfavorable reaction (positive $\Delta G$):

$$
Glucose + P_i \rightarrow Glucose\!-\!6\!-\!phosphate + H_2O
$$

2. Favorable ATP hydrolysis:

$$
ATP + H_2O \rightarrow ADP + P_i
$$

3. In cells, the enzyme hexokinase catalyzes the coupled reaction:

$$
Glucose + ATP \rightarrow Glucose\!-\!6\!-\!phosphate + ADP
$$

The enzyme’s active site binds both substrates and transfers the terminal phosphate of ATP directly to glucose. The sum of the two processes has $\Delta G < 0$, so the overall reaction proceeds.

Similar coupling principles apply broadly to:

• Mechanical work (muscle contraction, motor proteins)
• Active transport (ion pumps)
• Biosynthetic reactions (peptide bond formation, nucleic acid polymerization)

Other Energy Carriers and Redox Coenzymes

Besides ATP, metabolism relies heavily on redox coenzymes:

• $NAD^+ / NADH$: mainly catabolic, involved in oxidative pathways.
• $NADP^+ / NADPH$: mainly anabolic, provides reducing power for biosynthesis.
• $FAD / FADH_2$: tightly bound to enzymes, participates in various oxidation reactions.
• Coenzyme A (CoA): carries acyl groups (e.g. acetyl–CoA) rather than electrons, but is central in energy metabolism.

In many catabolic reactions, nutrients are oxidized and coenzymes like $NAD^+$ are reduced to $NADH$. Electrons from $NADH$ are later transferred to oxygen (aerobic organisms) via respiratory chains, generating ATP.

Biocatalysis: Enzymes as Biological Catalysts

General Characteristics of Enzymes

Enzymes are mostly proteins (some RNAs act as catalysts and are called ribozymes) that:

• Increase the rate of reactions by lowering the activation energy ($E_a$), without altering $\Delta G$ or the equilibrium position.
• Operate under mild conditions: neutral pH, moderate temperature, aqueous environment.
• Show high specificity for their substrates and the reactions they catalyze.
• Are not consumed in the reaction; they can turn over many substrate molecules.

Conceptual energy diagram for a reaction:

• Uncatalyzed: high $E_a$ → slow rate.
• Enzyme‑catalyzed: lower $E_a$ via an alternative reaction pathway → fast rate.

Active Site, Substrate Binding, and Specificity

The active site is the region of an enzyme where substrate binding and catalysis occur:

• Usually a three‑dimensional pocket or groove formed by amino acid side chains.
• Often contains key residues that donate/accept protons, form temporary covalent bonds, or stabilize charged intermediates.

Models of substrate binding:

• Lock‑and‑key: enzyme and substrate fit together with shape complementarity.
• Induced fit: binding of the substrate induces a conformational change in the enzyme, optimizing interactions with the transition state and increasing catalytic efficiency.

Types of specificity:

• Substrate specificity: recognizes a particular substrate or a class of structurally related substrates (e.g. proteases that cleave specific peptide bonds).
• Stereospecificity: distinguishes between stereoisomers, e.g. enzymes that act only on L‑amino acids or D‑sugars.

Catalytic Mechanisms (Conceptual)

Enzymes employ a variety of catalytic strategies (detailed mechanisms are treated in more advanced courses). Conceptually:

• Acid–base catalysis: amino acid side chains donate or accept protons to stabilize transition states.
• Covalent catalysis: enzyme forms a short‑lived covalent intermediate with the substrate, which then breaks down to product.
• Metal ion catalysis: bound metal ions help orient substrates, stabilize charges, or participate in redox reactions.
• Proximity and orientation effects: substrates are brought together at the right orientation and distance, increasing effective concentration and reaction rate.
• Transition state stabilization: the active site binds the transition state more tightly than the substrate, lowering activation energy.

Coenzymes and Prosthetic Groups

Many enzymes require additional non‑protein components for activity:

• Cofactors: non‑protein components required for catalysis, which can be:
• Metal ions: e.g. $Mg^{2+}$, $Zn^{2+}$, $Fe^{2+}/Fe^{3+}$.
• Coenzymes: small organic molecules (often derived from vitamins), e.g. $NAD^+$, FAD, coenzyme A, biotin.
• Prosthetic groups: tightly or covalently bound cofactors (e.g. heme in cytochromes).

Coenzymes often participate directly in chemical transformations (e.g. electron transfer, group transfer) and are regenerated in subsequent steps.

Enzyme Kinetics (Qualitative)

The relationship between substrate concentration and reaction rate follows characteristic patterns; one of the most important is captured by the Michaelis–Menten framework:

• At low substrate concentration, rate increases almost linearly with [S].
• At high substrate concentration, the enzyme becomes saturated and the rate approaches a maximum ($V_{max}$).

A key parameter is $K_M$ (Michaelis constant):

• Roughly reflects the substrate concentration at which the reaction rate is half‑maximal.
• Gives a measure of the enzyme’s affinity for the substrate (lower $K_M$ → higher apparent affinity).

You do not need the full kinetic equations here; the essential idea is:

• Enzymes have limited capacity (due to saturation), and their activity depends on substrate concentration and on regulatory influences.

Regulation of Metabolism and Enzyme Activity

Cells must carefully coordinate catabolic and anabolic pathways. Regulations occur at multiple levels.

Allosteric Regulation

Many enzymes are controlled by allosteric effectors:

• An allosteric effector binds at a site distinct from the active site (the allosteric site).
• Binding alters the enzyme’s conformation, changing its activity (activation or inhibition).

Common patterns:

• Feedback inhibition: the end product of a metabolic pathway inhibits an enzyme that acts early in that same pathway. This prevents wasteful overproduction.
• Feed‑forward activation: a metabolite early in a pathway may activate enzymes downstream, preparing the pathway for increased flux.

Allosteric enzymes often show sigmoidal (S‑shaped) dependence of reaction rate on substrate concentration, reflecting cooperative behavior (binding of one substrate molecule affects binding of others).

Covalent Modification

Enzymes can be reversibly activated or inactivated by covalent modifications, such as:

• Phosphorylation and dephosphorylation:
• Protein kinases transfer a phosphate group from ATP to specific amino acid side chains (often serine, threonine, or tyrosine).
• Phosphatases remove these phosphate groups.
• Depending on the enzyme, phosphorylation can either increase or decrease activity.

Other modifications include acetylation, methylation, and ubiquitination, often serving regulatory or signaling roles.

Control of Enzyme Amount

Over longer timescales, cells adjust how much of an enzyme is present:

• Regulation at the levels of gene transcription, mRNA stability, translation, and protein degradation.
• For example, when a particular nutrient is abundant, enzymes that metabolize that nutrient may be expressed at higher levels.

This kind of regulation is slower but can have large and lasting effects on metabolic capacities.

Compartmentation and Channeling

Compartmentation contributes to regulation by:

• Separating potentially incompatible reactions (e.g. synthetic and degradative pathways).
• Creating specialized microenvironments (pH, cofactor concentrations).

In some cases, enzymes of a pathway form multienzyme complexes or are arranged in a way that allows substrate channeling: intermediates pass directly from one active site to the next without diffusing into the bulk solution, increasing efficiency and preventing side reactions.

Central Examples of Metabolic Pathways (Conceptual)

Here we illustrate how metabolism and biocatalysis work together using a few central pathways. Detailed structures of carbohydrates, lipids, and amino acids are treated in other chapters; here we emphasize flow of matter and energy and the role of enzymes.

Glycolysis: A Central Catabolic Pathway

Glycolysis is a ten‑step pathway that converts one molecule of glucose into two molecules of pyruvate, with the net production of ATP and $NADH$.

• Occurs in the cytosol.
• Can operate with or without oxygen (aerobic vs anaerobic conditions).

Conceptually divided into two phases:

1. Investment phase:
• ATP is consumed to phosphorylate glucose and intermediates.
• Phosphorylation “tags” glucose for metabolism and traps it in the cell.
2. Payoff phase:
• ATP is produced by substrate‑level phosphorylation (direct transfer of phosphate from a high‑energy intermediate to ADP).
• $NAD^+$ is reduced to $NADH$.

Key ideas:

• Several steps have large negative $\Delta G$ and are catalyzed by regulatory enzymes, making them control points for glycolysis.
• In the absence of oxygen, cells regenerate $NAD^+$ via fermentation (e.g. lactate or ethanol formation), which allows glycolysis to continue.

Citric Acid Cycle and Oxidative Phosphorylation (Overview)

In aerobic organisms:

• Pyruvate (from glycolysis) is converted to acetyl–CoA.
• Acetyl–CoA enters the citric acid cycle (in mitochondria in eukaryotes), where:
• The acetyl group is fully oxidized to $CO_2$.
• A series of enzyme‑catalyzed reactions generate $NADH$, $FADH_2$, and a small amount of GTP or ATP.

Electrons from $NADH$ and $FADH_2$ are then transferred to oxygen via the electron transport chain, generating a proton gradient across a membrane. ATP synthase, another enzyme complex, uses this gradient to synthesize ATP from ADP and $P_i$ (oxidative phosphorylation).

Key points:

• Stepwise enzymatic oxidation allows controlled energy capture.
• The citric acid cycle also provides intermediates for biosynthetic pathways (e.g. amino acid and nucleotide synthesis).

Anabolic Pathways: Biosynthesis of Macromolecules

Anabolic pathways build macromolecules from simpler precursors:

• Protein synthesis (translation):
• Amino acids are activated by attachment to tRNA using ATP.
• The ribosome catalyzes peptide bond formation (a condensation reaction) in a templated, highly regulated manner.
• Fatty acid synthesis:
• Acetyl–CoA units are combined and reduced, using ATP and NADPH.
• Reactions are catalyzed by a multi‑enzyme complex (fatty acid synthase in many organisms).
• Polysaccharide synthesis:
• Monosaccharides are activated as sugar nucleotides (e.g. UDP‑glucose).
• Glycosyltransferases catalyze formation of glycosidic bonds.

All these processes are energy‑consuming and tightly regulated to ensure that biosynthesis aligns with nutrient availability and cellular needs.

Biocatalysis in Metabolic Pathways: Examples

Enzyme Cascades and Pathway Efficiency

Within a pathway, multiple enzymes act sequentially:

• Each step has a specific enzyme, ensuring high fidelity and minimizing side products.
• Many pathways are organized so that intermediates are promptly used, avoiding accumulation of potentially harmful compounds.
• Enzyme cascades can amplify small regulatory signals (common in signaling and hormone‑controlled pathways).

Example concept: In glycogen breakdown, a hormone signal (e.g. adrenaline) triggers a phosphorylation cascade, ultimately activating glycogen phosphorylase, which catalyzes the release of glucose units from glycogen.

Isoenzymes and Tissue Specialization

Isoenzymes (isozymes) are different molecular forms of an enzyme that catalyze the same reaction but differ in regulation or kinetics.

• Allow tissue‑specific control of metabolism.
• Example concept: different isoforms of lactate dehydrogenase in heart and skeletal muscle reflect different metabolic priorities (aerobic vs more anaerobic conditions).

Biocatalysis Beyond Metabolism: Biotechnological Use

The principles of metabolic biocatalysis are applied in:

• Industrial biocatalysis:
• Enzymes used to catalyze specific reactions in synthesis of chemicals, pharmaceuticals, and fine chemicals.
• Advantages: high selectivity, milder conditions, often more environmentally friendly (“green chemistry”).
• Metabolic engineering and synthetic biology:
• Redesigning metabolic pathways or introducing new enzymes in microorganisms to produce fuels, materials, or drugs.
• Requires detailed knowledge of metabolic networks and regulatory mechanisms.

Integration: Homeostasis and Metabolic Flexibility

Metabolism and biocatalysis together provide cells with:

• Homeostasis: maintaining relatively constant internal conditions despite changing external environment.
• Stable levels of ATP, key metabolites, and macromolecules.
• Flexibility: ability to alter fluxes through pathways depending on:
• Nutrient availability (e.g. switch between carbohydrate and fat utilization).
• Oxygen supply (aerobic vs anaerobic metabolism).
• Energy demand (rest vs intense activity).
• Developmental stage or environmental stress.

This integration is achieved by orchestrating:

• The activities of numerous enzymes (biocatalysts).
• The flows of energy (ATP, redox carriers).
• The interactions of pathways in a complex but coordinated metabolic network.

Understanding metabolism and biocatalysis provides the foundation for interpreting how organisms obtain and use energy, grow, adapt, and respond to their environment—and for manipulating these processes in medicine, biotechnology, and environmental applications.