Bioch lec 5- enzymes
What’s special about enzymes compared to industrial catalysts?
They dramatically accelerate reactions under biological conditions (ambient temperature, atmospheric pressure), unlike industrial processes that may need extreme heat/pressure (e.g., Haber process)
What do enzymes do in metabolism (e.g., extracting energy from glucose)?
Enzymes allow controlled, stepwise oxidation of glucose to capture energy efficiently rather than explosive release (e.g., burning fuels).
What are enzymes?
Biological catalysts that speed up biochemical reactions without being consumed. Most are globular proteins with defined 1°, 2°, 3°, and sometimes 4° structure
Two ways to accelerate a chemical reaction
Add heat (increases number of molecules overcoming activation barrier).
Add a catalyst (lowers activation energy without being consumed).
Enzymes use method #2.
Why are enzymes proteins?
Their function depends on flexible, complex 3-D structure driven by the same forces governing protein folding.
Four key properties of enzymes as catalysts
Increase reaction rates (10⁶–10²⁰×).
Are regenerated at the end.
Highly specific (no side reactions).
Promote conversion A + B → C + D.
Enzyme nomenclature: what does “-ase” indicate?
The name usually reflects substrate (e.g., alcohol dehydrogenase), product, or reaction type (e.g., dehydrogenase, decarboxylase).
Does a negative ΔG guarantee a fast reaction?
No. Thermodynamically favorable reactions can still be slow if the activation energy barrier is large. ΔG relates to spontaneity, not rate
What determines reaction speed in biochemistry?
The size of the activation energy (ΔG‡) barrier between reactants and the transition state. Smaller barrier → faster reaction.
What do enzymes change in a reaction?
Enzymes lower ΔG‡ by stabilizing the transition state. They do NOT change ΔG of the overall reaction
Four major ways enzymes reduce ΔG‡
Desolvation (removing substrates from water).
Proximity and orientation effects.
Participation in reaction chemistry (acid/base, covalent, metal).
Transition state stabilization.
What is an active site?
A 3-D cleft where catalysis occurs; contains key residues for binding & reaction; determines specificity, affinity, and rate. Complementary to substrate/TS.
Lock-and-Key vs. Induced Fit (basic distinction)
Lock-and-Key: Rigid complementarity.
Induced Fit: Substrate binding causes enzyme to change shape → better binding + catalytic alignment.
Three advantages of excluding water from the active site
Removes water shell → faster reaction.
Strengthens polar interactions (H-bonds, ion pairs).
Prevents side reactions.
What happens during induced fit?
Enzyme conformational change: closes active site, excludes more water, and positions catalytic residues optimally. Example: hexokinase.
Proximity & orientation—why they matter
Reactions only occur if substrates collide in the correct geometry. Active sites hold substrates close and properly oriented → ~1000-fold rate boost.
Do all enzymes use proximity & orientation effects?
Yes, all enzymes use this mechanism to some degree.
Three major chemical catalytic strategies enzymes use
Acid/base catalysis
Covalent (nucleophilic) catalysis
Metal ion catalysis
May be via amino acids or cofactors.
What amino acid side chains can act as acid/base catalysts?
Any residue capable of donating/accepting protons depending on protonation state (e.g., His, Asp, Glu, Lys, Tyr).
What amino acids commonly serve as nucleophiles?
Ser, Cys, Lys, His, Asp, Glu — often activated by deprotonation.
Cofactors: three classes
Metal ions (Fe, Zn, Mg, etc.)
Coenzymes (organic molecules)
Cosubstrates (loosely bound; e.g., NAD⁺)
Prosthetic groups (tightly bound; e.g., FAD)
These expand reactive capability.
Apoenzyme vs. holoenzyme
Apoenzyme: Inactive protein without its prosthetic group.
Holoenzyme: Active enzyme with prosthetic group attached.
Same for apoprotein/holoprotein.
Transition state stabilization: why is it powerful?
The enzyme binds the transition state more tightly than substrate → lowers ΔG‡ → increases rate dramatically.
Why are transition state analogs potent inhibitors?
They mimic TS, which enzymes bind with highest affinity → very strong competitive inhibitors (basis for drug design).
Define V₀, Vmax, Km
V₀: Initial rate of product formation.
Vmax: Maximum achievable rate at saturating substrate.
Km: [S] at which V₀ = ½ Vmax; inverse indicator of affinity (low Km = high affinity).
What happens to reaction velocity as [S] increases?
Increases hyperbolically until approaching Vmax (Michaelis–Menten curve).
Which substrate has higher affinity? (Based on lower Km)
The substrate with the lower Km has the higher enzyme affinity.
6 mechanisms of enzyme regulation
Competitive inhibition
Allostery
Reversible covalent modification
Ionic signals (e.g., Ca²⁺)
Gene expression
Subcellular localization
Intrinsic vs. non-intrinsic activity modifiers.
What defines a competitive inhibitor?
Binds reversibly to the active site; resembles substrate or TS but does not react. Blocks substrate binding.
Effect of competitive inhibition on kinetics
Km increases (lower apparent affinity)
Vmax unchanged
Can be overcome by ↑[S]
Curves shift right.
Why do transition-state analogs outperform substrate analogs as competitive inhibitors?
Enzymes bind TS with greatest affinity, so TS-mimicking inhibitors bind extremely tightly
What characterizes allosteric enzymes?
Oligomeric, cooperative, sigmoidal kinetics; shift between T (tense, low activity) and R (relaxed, high activity) states.
Define homoallostery vs. heteroallostery
Homoallostery: Substrate itself enhances activity (positive cooperativity).
Heteroallostery: Other molecules (activators/inhibitors) modulate activity.
How do allosteric activators change enzyme behavior?
Shift equilibrium toward R state → increased activity → sigmoidal curve shifts left.
How do allosteric inhibitors affect activity?
Favor T state, decrease activity, sigmoidal curve shifts right.
Most common form of reversible covalent modification?
Phosphorylation of Ser/Thr/Tyr –OH groups.
How does phosphorylation alter enzyme activity?
Adds a bulky, charged phosphate → significantly alters tertiary structure → may activate or inactivate enzyme.
Enzymes that regulate phosphorylation
Protein kinases add phosphate (ATP → ADP).
Protein phosphatases remove phosphate (hydrolysis).
Both are regulated themselves.
Can residues besides Ser/Thr/Tyr be phosphorylated?
Yes (Asp, Glu, His), but these rarely participate in regulation (e.g., Asp in Na⁺/K⁺ ATPase).
Which regulatory mechanisms alter intrinsic catalytic activity?
Competitive inhibition, allostery, covalent modification.
Which do not alter intrinsic activity? Gene expression & localization.
Which mechanisms influence Km, Vmax, or both?
Competitive inhibitors: ↑Km, Vmax unchanged
Allosteric inhibitors: usually decrease Vmax & increase Km (varies)
Activators: decrease Km, may increase Vmax
Relationship between transition state stabilization and TS analog inhibition
The better an enzyme stabilizes the TS, the stronger a TS analog binds → explains their potency as drugs
Why do enzymes require structure flexibility?
Flexibility allows induced fit, TS stabilization, water exclusion, and regulation (e.g., phosphorylation). Static structures couldn't catalyze effectively.
Why does phosphorylation have such a dramatic effect?
Adds size, charge, and polarity, altering protein folding → modifies enzyme conformation allosterically.
In chymotrypsin, what is the role of the oxyanion hole and what level of detail do you need to remember?
Formed by backbone NH groups, it stabilizes the negatively charged oxygen that appears in the transition state (classic example of transition state stabilization).
You do not need to memorize the full mechanism or residue numbers, just the concept that it stabilizes the TS and lowers ΔG‡.