Lecture 3 Exam (Enzymes)

Active site

The specific region of an enzyme's 3D structure where substrate(s) bind non-covalently and catalysis occurs

How does substrate binding lower the energy barrier?

Binding releases the substrate's binding energy, which is used to help stabilize/reach the transition state and lower the activation energy (EA)

Activation energy (EA)

The energy difference between the transition state and the reactants; enzymes lower EA to speed up the reaction

Exergonic vs endergonic reaction

Exergonic: products have less energy than reactants, releases energy, spontaneous (ΔG negative, ΔH negative). Endergonic: products have more energy, consumes energy, non-spontaneous (ΔG positive, ΔH positive)

Example of a highly exergonic reaction in metabolism

Citrate synthase reaction (acetyl-CoA + oxaloacetate → citrate), ΔG = -32 kJ/mol

Keq (equilibrium constant)

The ratio of products to substrates at equilibrium; enzymes do not change Keq, only the speed of reaching equilibrium

Relationship between Keq and ΔG°'

Inverse/logarithmic: ΔG°' = -RT ln(Keq); more negative ΔG°' means larger Keq

Lock-and-key model

Older model of enzyme-substrate binding; assumes the enzyme is a fixed structure complementary in shape to the substrate

Induced fit model

Current model; substrate binding causes a conformational change in the enzyme so the active site becomes complementary to the substrate's shape

Clam-shell model example

Hexokinase — glucose binding causes conformational closure around the substrate, excluding water, before Mg-ATP binds and phosphorylation occurs

Role of Mg2+ in hexokinase and GAP dehydrogenase reactions

Shepherds/stabilizes the phosphate group during substrate-level phosphorylation, since phosphate carries a strong negative charge

Cofactor vs coenzyme

Cofactor = general helper molecule needed for enzyme function (often a metal ion: Zn2+, Mg2+, Mn, etc.); coenzyme = a specific type of cofactor, usually vitamin-derived (e.g. TPP, FAD, NAD, CoA, biotin)

Bronsted-Lowry acid/base definition

Acid donates H+; Base accepts H+

Lewis acid/base definition

Lewis acid accepts an electron pair; Lewis base donates an electron pair (broader than Bronsted-Lowry; biological Lewis acids are often cationic cofactors like Zn2+, Mg2+)

Electrophile vs nucleophile

Electrophile: positively charged/electron-poor, accepts electrons. Nucleophile: negatively charged/electron-rich, donates electrons

Covalent catalysis

Substrate forms a transient covalent bond with an active-site residue or cofactor, lowering the transition state energy; the bond must later be broken to regenerate the enzyme

Catalytic triad

Three active-site residues (typically Ser-His-Asp) that enable covalent catalysis, as in serine proteases like chymotrypsin and trypsin

Metal ion catalysis

Use of tightly or loosely bound metal ions (Fe, Cu, Zn, Na, K, Mg, Ca) to stabilize the transition state or unstable intermediates

Lysozyme's catalytic mechanism

Uses both acid-base and covalent catalysis to hydrolyze the glycosidic bond in bacterial peptidoglycan, adding water across the bond

Michaelis-Menten plot axes and key values

X-axis = substrate concentration, Y-axis = initial velocity; Vmax = maximum rate at enzyme saturation; KM = substrate concentration at ½Vmax (reflects enzyme-substrate affinity)

Lineweaver-Burk plot

Double-reciprocal plot (1/V vs 1/[S]); Y-intercept = 1/Vmax, X-intercept = -1/KM, slope = KM/Vmax; useful for characterizing inhibition

Hill plot

Log-log plot showing cooperativity of substrate binding (e.g. oxygen binding to hemoglobin); slope = Hill coefficient, higher value = greater cooperativity

kcat (turnover number)

kcat = Vmax/[E]T — the maximum number of substrate molecules converted to product per active site per second

kcat/KM

A measure of catalytic efficiency, valid when substrate concentration is much lower than KM

Competitive inhibition effect on Vmax and KM

Vmax unchanged, KM increases (apparent decrease in substrate affinity); inhibitor competes with substrate for the active site

Non-competitive inhibition effect on Vmax and KM

Vmax decreases, KM unchanged; inhibitor binds enzyme regardless of substrate binding, causing a conformational change

Allosteric protein / cooperativity

A protein (enzyme or transport protein) that changes conformation when a ligand binds one subunit, affecting affinity/activity of other subunits (e.g. hemoglobin, ATCase)