Low Km = high affinity. High Km = low affinity. Km = [S] at ½ Vmax.
Michaelis Constant (Km)
Km tells you how tightly the enzyme binds its substrate
Km = substrate concentration at half-maximal velocity. Low Km: enzyme achieves half-Vmax at low substrate → tight binding, high affinity. Km doesn't change with enzyme concentration.
Competitive Inhibition
Competitive inhibition: same active site, Km increases, Vmax unchanged — overcome with substrate
Competitive Inhibition
Inhibitor competes with substrate for the active site
Add more substrate → outcompete inhibitor → Vmax restored. Km appears to increase. Example: methotrexate competes with folate at DHFR (dihydrofolate reductase).
Non-Competitive Inhibition
Non-competitive: binds elsewhere, Vmax decreases, Km unchanged — can't overcome
Non-Competitive Inhibition
Inhibitor binds allosteric site — more substrate won't help
Inhibitor binds separate (allosteric) site, changes enzyme shape. Vmax decreases, Km unchanged. Adding more substrate doesn't help. Example: heavy metal ions (lead, mercury) inhibit enzymes non-competitively by binding to sulfhydryl groups away from the active site.
Cofactors and Coenzymes
Cofactors = metal ions. Coenzymes = organic (often from vitamins).
Cofactors and Coenzymes
Non-protein helpers that many enzymes require to function
Metal cofactors: Zn²⁺ (carbonic anhydrase), Fe²⁺ (cytochrome), Mg²⁺ (kinases). Coenzymes: NAD⁺, FAD, CoA — many derived from B vitamins. Deficiency → enzyme dysfunction.
Allosteric Regulation
Allosteric regulation: effector binds non-active site → changes enzyme shape → activates or inhibits
Allosteric Regulation
Enzymes can be turned on or off by molecules binding away from the active site
Positive allosteric effectors: bind and increase activity. Negative effectors: decrease activity. Feedback inhibition: product of a pathway inhibits an early enzyme — classic regulation strategy.
Six Enzyme Classes
Enzyme classification: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases — OT HaLIL (O=Oxidoreductases, T=Transferases, H=Hydrolases, L=Lyases, I=Isomerases, L=Ligases)
Six Enzyme Classes
The six classes of enzymes classified by the reaction they catalyze
Oxidoreductases: catalyze oxidation-reduction. Transferases: transfer functional groups. Hydrolases: cleave bonds with water (proteases, lipases). Lyases: cleave bonds without water (non-hydrolytic). Isomerases: convert isomers. Ligases: join two molecules using ATP. Most drugs target enzymes in these classes.
Oxidoreductases
Oxidation-reduction reactions
Transferases
Transfer functional groups
Hydrolases
Cleave with water
Lyases
Cleave without water
Isomerases
Convert between isomers
Ligases
Join molecules using ATP
Activation Energy
Activation energy: energy barrier a reaction must overcome. Enzymes lower it — don't change ΔG.
Activation Energy
What enzymes actually do — and what they don't change
Activation energy (Ea): energy needed to start a reaction. High Ea → slow reaction. Enzymes provide an alternative pathway with lower Ea → faster reaction. Crucially: enzymes do NOT change the equilibrium constant (K_eq) or the overall free energy change (ΔG). They speed up reactions that would happen anyway.
Feedback Inhibition
Feedback inhibition: the END product of a pathway inhibits an EARLY enzyme — classic metabolic control
Feedback Inhibition
How cells regulate metabolic pathways through end-product inhibition
Isoleucine synthesis: threonine → (5 steps) → isoleucine. When isoleucine accumulates, it inhibits the first enzyme in the pathway (allosterically). Efficient: stops the whole pathway when product is abundant. Avoids wasteful overproduction. Classic example of negative feedback in biochemistry.
Prosthetic groups: cofactors permanently attached to enzyme. Heme in hemoglobin and cytochrome c.
Prosthetic Groups
Permanently bound cofactors essential for enzyme function
Prosthetic group: non-protein component permanently and tightly bound to the protein. Unlike coenzymes (loosely bound, can leave). Heme group: iron-containing porphyrin ring — in hemoglobin (O₂ transport), myoglobin, cytochromes (ETC). FAD: covalently bound in some enzymes. Biotin: covalently bound in carboxylases.
Zymogens protect cells from premature enzymatic activity. Digestive enzymes stored as zymogens in pancreas — activated only in the intestine. Pepsinogen (stomach) → pepsin (activated by stomach acid). Trypsinogen → trypsin (activated by enteropeptidase in small intestine). Blood clotting cascade: sequential zymogen activation.
Mnemonic
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🎓 Common Exam Questions
Q: Explain Michaelis-Menten kinetics — what do Km and Vmax tell you?
A: The Michaelis-Menten equation: V = Vmax[S] / (Km + [S]). Vmax is the maximum reaction velocity when all enzyme is saturated with substrate — proportional to enzyme concentration. Km is the substrate concentration at which velocity = ½ Vmax. Km approximates the enzyme-substrate dissociation constant — low Km = high affinity (enzyme is half-saturated at low [S]); high Km = low affinity. At low [S], V is proportional to [S] (first-order kinetics). At high [S], V approaches Vmax (zero-order kinetics). Km and Vmax are determined experimentally from the Michaelis-Menten curve or Lineweaver-Burk double-reciprocal plot.
Q: Compare competitive and non-competitive enzyme inhibition.
A: Competitive inhibition: inhibitor resembles substrate, binds active site, competes directly with substrate. Effect: Km increases (more substrate needed to displace inhibitor); Vmax unchanged (can be overcome by excess substrate). Example: statins competitively inhibit HMG-CoA reductase. Non-competitive inhibition: inhibitor binds allosteric site (not active site), changes enzyme conformation. Effect: Vmax decreases (enzyme is less effective even when substrate-bound); Km unchanged (affinity unaffected). Cannot be overcome by adding substrate. Example: cyanide inhibiting cytochrome c oxidase. On Lineweaver-Burk: competitive changes x-intercept; non-competitive changes y-intercept.
Q: What is allosteric regulation and why is it important in metabolism?
A: Allosteric regulation occurs when a molecule (effector) binds at a site other than the active site (allosteric site), inducing a conformational change that activates or inhibits the enzyme. Allosteric enzymes typically show sigmoidal (not hyperbolic) kinetics — cooperativity. Importance in metabolism: allosteric enzymes are usually at committed steps of metabolic pathways; they sense metabolic status and respond instantly. Example: phosphofructokinase-1 (PFK-1) in glycolysis is allosterically inhibited by ATP and citrate (energy surplus) and activated by AMP and ADP (energy deficit). This allows cells to regulate energy production in real time without changing gene expression.
Q: What are zymogens and why are they important?
A: Zymogens (proenzymes) are inactive enzyme precursors activated by proteolytic cleavage — removing an inhibitory peptide fragment. This is a crucial safety mechanism: digestive proteases (pepsinogen → pepsin, trypsinogen → trypsin, chymotrypsinogen → chymotrypsin) are made in inactive form to prevent self-digestion of the cells that produce them. Activation occurs only in the appropriate location (stomach lumen, small intestine). Blood clotting cascade: clotting factors are zymogens activated in sequence — a cascade amplification system enabling rapid, localized clot formation. Premature zymogen activation causes pancreatitis (trypsinogen activated within the pancreas).
Q: What is feedback inhibition and give a biochemical example?
A: Feedback inhibition is when the end product of a metabolic pathway allosterically inhibits an early (usually the first committed step) enzyme in that pathway — preventing overproduction. Classic example: in pyrimidine biosynthesis, CTP (end product) inhibits ATCase (aspartate transcarbamoylase) — the first committed step enzyme. In amino acid biosynthesis: isoleucine inhibits threonine deaminase (first step of isoleucine synthesis). In glycolysis: ATP and citrate inhibit PFK-1. This elegant regulatory mechanism is energy-efficient, rapid (seconds), and sensitive — the cell produces exactly what it needs without wasting resources.