Mechanisms and Inhibitors

Vocabulary practice flashcards covering catalytic strategies, temperature and pH effects, reversible and irreversible enzyme inhibition mechanisms, Lineweaver–Burk interpretations, and the catalytic mechanism of chymotrypsin.

Covalent Catalysis

A catalytic strategy in which the enzyme's active site contains a reactive group—usually a powerful nucleophile—that becomes temporarily covalently modified during catalysis.

Nucleophile

A chemical group or atom that is attracted to regions of positive charge in other molecules and participates in chemical reactions by donating electrons to electrophiles.

Electrophile

An electron-deficient chemical group that is attracted to negative charge and participates in chemical reactions by accepting electrons from nucleophiles.

General Acid–Base Catalysis

A catalytic mechanism in which a molecule other than water acts as a proton donor or proton acceptor, such as histidine in chymotrypsin enhancing the nucleophilic power of serine.

Metal Ion Catalysis

A catalytic strategy where bound metal ions act as electrophilic catalysts to stabilize intermediate negative charges, generate nucleophiles by increasing the acidity of nearby molecules, or increase binding energy.

Catalysis by Approximation

A catalytic enhancement in reactions with multiple substrates that occurs by bringing the substrates into proximity and proper orientation on a single enzyme binding surface.

Binding Energy

The free energy released in enzyme–substrate interactions that reaches its maximum when the enzyme binds the transition state, favoring transition-state formation and promoting catalysis.

Thermal Denaturation

The abrupt loss of enzyme structure and catalytic activity when temperature increases past an optimum, causing thermal jostling that disrupts noncovalent interactions maintaining 3D conformation.

Endotherms

Organisms, such as humans, that maintain a constant body temperature, thereby minimizing the impact of ambient temperature fluctuations on enzyme activity.

Ectotherms

Organisms that assume the temperature of their ambient environment, causing temperature to act as a direct regulator of their biochemical and biological activity.

Optimal pH

The specific $pH$ at which an enzyme displays maximal catalytic activity, reflecting the ionization of active-site residues and the physiological environment of the enzyme.

Irreversible Inhibitor

An inhibitor that covalently binds to a target enzyme and does not dissociate at an appreciable rate, permanently halting enzyme activity.

Reversible Inhibitor

An inhibitor that binds to an enzyme predominantly via noncovalent interactions and is characterized by rapid dissociation of the enzyme–inhibitor complex.

Competitive Inhibition

A form of reversible inhibition where the inhibitor competes with substrate for the active site, increasing the apparent value of $K_M$ ($K_M^{\text{app}}$) without altering $V_{\max}$.

Competitive Inhibition (Lineweaver–Burk Plot)

A double-reciprocal plot where the inhibited enzyme line shares an identical y-intercept ($1/V_{\max}$) with the uninhibited line, but exhibits an increased slope ($K_M/V_{\max}$).

Uncompetitive Inhibition

A form of reversible inhibition where the inhibitor binds exclusively to the enzyme–substrate ($ES$) complex, lowering both apparent $V_{\max}$ ($V_{\max}^{\text{app}}$) and apparent $K_M$ ($K_M^{\text{app}}$).

Uncompetitive Inhibition (Lineweaver–Burk Plot)

A double-reciprocal plot characterized by parallel lines between uninhibited and inhibited enzymes, displaying identical slopes ($K_M/V_{\max}$) and an increased y-intercept ($1/V_{\max}^{\text{app}}$).

Pure Noncompetitive Inhibition

A reversible inhibition mechanism in which the inhibitor binds equally well to free enzyme ($E$) and the $ES$ complex, decreasing $V_{\max}$ to $V_{\max}^{\text{app}}$ while leaving $K_M$ unchanged.

Pure Noncompetitive Inhibition (Lineweaver–Burk Plot)

A double-reciprocal plot where the uninhibited and inhibited enzyme lines intersect directly on the x-axis ($-1/K_M$ is identical), while the inhibited line has a greater y-intercept and steeper slope.

Mixed Noncompetitive Inhibition

A reversible inhibition mechanism in which the inhibitor binds preferentially to either free enzyme ($E$) or the $ES$ complex, decreasing $V_{\max}$ to $V_{\max}^{\text{app}}$ and either increasing or decreasing $K_M$.

Transition-State Analog (TSA)

A stable compound synthesized to mimic the transition state of a catalyzed reaction, functioning as a highly potent and specific competitive inhibitor due to selective active-site binding.

Group-Specific Reagents

A class of irreversible inhibitors that covalently modify specific $R$ groups of amino acids throughout an enzyme.

Diisopropylphosphofluoridate (DIPF)

A group-specific irreversible inhibitor that modifies a single critical serine residue out of $28$ in chymotrypsin, and also inactivates acetylcholinesterase.

Affinity Labels (Reactive Substrate Analogs)

Molecules structurally similar to an enzyme's substrate that covalently bind to active-site residues, providing greater active-site specificity than group-specific reagents.

Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK)

An affinity label for chymotrypsin containing a phenylalanine moiety that binds the active site and a chloromethyl ketone group that irreversibly alkylates histidine $57$.

Mechanism-Based (Suicide) Inhibitors

Chemically modified substrates that bind to an enzyme's active site and undergo the initial catalytic steps, generating a reactive intermediate that covalently inactivates the enzyme.

Penicillin

A suicide inhibitor antibiotic consisting of a thiazolidine ring fused to an unstable, reactive four-membered $\beta$-lactam ring that mimics a $\text{D-Ala-D-Ala}$ peptide.

Peptidoglycan

A bacterial cell wall macromolecule consisting of linear polysaccharide chains cross-linked by short peptides, conferring mechanical stability against internal osmotic pressure.

Glycopeptide Transpeptidase

A bacterial enzyme that catalyzes peptide cross-link formation in peptidoglycan synthesis; it is permanently inactivated by penicillin forming a covalent penicilloyl-enzyme complex.

Christopher T. Walsh

A biochemist known for unravelling enzymatic reaction mechanisms, suicide inhibition, antibiotic actions, and microbial natural product synthesis.

Proteolytic Enzymes (Proteases)

A large class of enzymes that catalyze the breakdown of proteins into smaller peptides and amino acids through peptide bond hydrolysis.

Chymotrypsin

A digestive protease that cleaves peptide bonds selectively on the carboxyl-terminal side of large hydrophobic amino acids (such as Trp, Tyr, Phe, Met, and Ile) via covalent catalysis.

Chromogenic Substrate

A synthetic substrate analog that produces a colored product upon enzymatic cleavage, enabling reaction kinetics to be tracked spectrophotometrically.

Burst Phase

The rapid initial generation of product observed in chymotrypsin pre-steady-state kinetics, representing stoichiometric acylation before the slower deacylation step limits the steady state.

Acyl-Enzyme Intermediate

A covalent intermediate formed during chymotrypsin catalysis where the acyl group of the substrate is linked to the side chain of serine $195$.

Catalytic Triad

The constellation of aspartate $102$, histidine $57$, and serine $195$ in chymotrypsin's active site that converts serine into a potent nucleophile.

Histidine 57

A catalytic triad residue that acts as a general base catalyst to deprotonate serine $195$, generating an alkoxide ion, and later deprotonates water during deacylation.

Aspartate 102

A catalytic triad residue that orients histidine $57$ and enhances its proton-accepting ability through hydrogen bonding and electrostatic effects.

Oxyanion Hole

A pocket in chymotrypsin that stabilizes the negatively charged oxygen atom of the tetrahedral intermediate via hydrogen bonds with the backbone $-NH-$ groups of glycine $193$ and serine $195$.

S1 Pocket

A deep, hydrophobic binding cleft in chymotrypsin that accommodates the long, uncharged hydrophobic side chains of substrates to correctly position the adjacent scissile bond.