Enzyme tricks that make kinetics click

Low Km = high affinity. High Km = low affinity. Km = [S] at ½ Vmax.

Michaelis Constant (Km) — 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: same active site, Km increases, Vmax unchanged — overcome with substrate

Competitive Inhibition — Add more substrate → outcompete inhibitor → Vmax restored. Km appears to increase. Example: methotrexate competes with folate at DHFR (dihydrofolate reductase).

Non-competitive: binds elsewhere, Vmax decreases, Km unchanged — can't overcome

Non-Competitive Inhibition — 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 = metal ions. Coenzymes = organic (often from vitamins).

Cofactors and Coenzymes — Metal cofactors: Zn²⁺ (carbonic anhydrase), Fe²⁺ (cytochrome), Mg²⁺ (kinases). Coenzymes: NAD⁺, FAD, CoA — many derived from B vitamins. Deficiency → enzyme dysfunction.

Allosteric regulation: effector binds non-active site → changes enzyme shape → activates or inhibits

Allosteric Regulation — Positive allosteric effectors: bind and increase activity. Negative effectors: decrease activity. Feedback inhibition: product of a pathway inhibits an early enzyme — classic regulation strategy.

Enzyme classification: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases — OT HaLIL (O=Oxidoreductases, T=Transferases, H=Hydrolases, L=Lyases, I=Isomerases, L=Ligases)

OxidoreductasesOxidation-reduction reactions

TransferasesTransfer functional groups

HydrolasesCleave with water

LyasesCleave without water

IsomerasesConvert between isomers

LigasesJoin molecules using ATP

Activation energy: energy barrier a reaction must overcome. Enzymes lower it — don't change ΔG.

Activation Energy — 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: the END product of a pathway inhibits an EARLY enzyme — classic metabolic control

Feedback 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.

Irreversible inhibitors: permanently inactivate enzyme by covalent bond. Aspirin, nerve agents, penicillin.

Irreversible Inhibitors — Irreversible inhibitors form covalent bonds with the enzyme — permanent inactivation. Aspirin: acetylates COX enzyme → blocks prostaglandin synthesis → anti-inflammatory, anti-platelet. Nerve agents (sarin): covalently inhibit acetylcholinesterase → nerve signals can't stop. Penicillin: covalently inhibits transpeptidase → bacterial cell wall synthesis stops.

Lineweaver-Burk plot: double reciprocal plot (1/V vs 1/[S]). Intercepts give Vmax and Km.

Lineweaver-Burk Plot — Plot 1/V (y-axis) vs 1/[S] (x-axis) → straight line. Y-intercept = 1/Vmax. X-intercept = -1/Km. Slope = Km/Vmax. Competitive inhibitor: increases slope (higher Km), same y-intercept (same Vmax). Non-competitive: same x-intercept (same Km), increases y-intercept (lower Vmax).

Prosthetic groups: cofactors permanently attached to enzyme. Heme in hemoglobin and cytochrome c.

Prosthetic Groups — 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.

Zymogen (proenzyme): inactive enzyme precursor activated by cleavage. Pepsinogen → pepsin, trypsinogen → trypsin.

Zymogens — 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.