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- biological catalysts, increase the rates of chemical rxn by lowering Ea (energy req’d to produce transition state), net energy Δ is the same, no change in thermodynamics, no change in Keq

- bind to substrates converting them into products

- return to their original form after rxn

- Conjugated proteins; apoenzyme – protein part; cofactor/prosthetic group– non-protein part, maybe ions (inorganic) or vitamin derivative (organic), Holoenzyme = apoenzyme + cofactor

- Varying sizes and weights, 10kd to 1000 kd

- Single or multiple subunits

- Very specific, 1 substrate = 1 product, no side reactions or byproducts

- Very efficient and effective, rxn rates by 10^5 – 10^12 fold

- Regulates metabolic rxn, by changing state of activity

Classification and Nomenclature

- -ase suffix to root word of substrate or trivial names

- 6 Major Classes


Class 1 – Oxidoreductases – redux rxn


Class 2 – Transferases – transfer chemical groups


Class 3 – Hydrolases – bond cleaving with water


Class 4 – Lyases – nonhydrolytic splitting of molecules or addition of groups to DBs


Class 5 – Isomerases – interconvert isomeric molecules


Class 6 – Ligases – create chem bonds at the expense of NTP

- Enzyme Commission Number: (name) general class. Subclass. Sub-subclass. Complete name

- Based on level of organization or # of subunits: monomeric, oligomeric, multienzyme

- Based on degree of presence in cell: constitutive (constant amount), or inducible (induced by

substrate, genetically controlled)

Enzyme Specificity and Efficiency

- specificity – active / catalytic site

- Active site – small part, binds with substrates, contains AA residues participating in bond making or breaking, 3D, multiple weak attractions, clefts or crevices or cavities, precision of arrangement of atoms

- Rigid Template (Key Lock) – substrate is complementary in structure to active site

- Induced Fit (Flexible) – as substrate binds, enzyme undergoes conformational change

Mechanisms of Enzymatic Catalysis

- Proximity and Orientation effects – reacting groups are close enough and properly oriented, favorable overlap of orbitals

- Desolvation effects – water is removed, creating hydrophobic environment, accelerating rxn

- Acid Base Catalysis – substrate complements enzyme, AA chains act as (+) donors and acceptors

- Covalent catalysis – nucleophilic AA attack electrophilic substrate = covalent bonds, holds substrate

- Strain Effects or Bond Distortion –conformational change distort some parts of the substrate

- Metal Coordination effects – metal ions electron pairs (Lewis acid) to form Σ bond, stabilizes negative change, favoring nucleophilic attack on substrate, may also form coordination bonds accelerating rxn

Favors affecting Enzyme Activity

- pH – bell shaped curve, may denaturate enzymes

- temperature – bell shaped curve, denatured enzymes at T

- enzyme concentration – proportional, linear, no effect on Keq

- substrate concentration – rectangular hyperbola, first order, zero order at Vmax

- Inhibitors irreversible (tightly bound to enzyme, loss of function or reversible


Competitive – resembles substrate, competes for active site, overcome by substrate conc


Non-competitive – binds to allosteric site, deactivating enzyme before binding


Uncompetitive – acts in ES complex, alters turnover rate

allosteric site, deactivating enzyme before binding o Uncompetitive – acts in ES complex, alters turnover rate

Enzyme Kinetics

- Units – Specific activity (µmol/min per mg protein), Turnover Number or Catalytic constant (Kcat, µmol/min per µmol enzyme), Katal (kat, amount of enzyme acitivty that transforms 1 mole of substrate per second)

- Assumptions of Michaelis and Menten


Enyzme reacts reversible, forms ES complex


[S]>>[E], possible to saturate E as ES with excess S


Product P does not accumulate appreciably, ES ! E+P neglible (K3<<K2)

- Steady State Assumption of Briggs and Haldane – ES is constant during initial velocity because ES formation = ES breakdown

- Derivation of Michaelis Menten Equation


Total enzyme conc Et = free enzyme E + bound enzyme ES


Michaelis Menten Equation: V = Vmax [S] / Km + [S]

- Significance of Km and Vmax


Km = [S] at ½ Vmax


measures affinity of an enzyme to substrate: Km = weak binding, Km = strong binding


Vmax – reveals turnover number (Vmax = k3[Et])

- Graphing


Michaelis Menten Graph – rectangular hyperbola, ½ Vmax, Vmax, km


Lineweaver Burk Equation – reciprocal of MME, linear slope, y intercept = 1/Vmax, x = 1/Km, slope = Km/Vmax


Eadie Hofstee plot – v=-Km x v/[S] + Vmax, slope = -Km

- Effects of Inhibition on LBE graph:









- Multisubstrate Kinetics


> 1 substrate – interactions may be bi-uni (2S, 1P) or bi-bi (2S, 2P)


Cleland notation system – illustrates rxn categories


Sequential Rxn – all S react first with enzyme before P release

" Ordered Sequential – obligatory order of addition of S and release of P

" Random Sequential – no obligatory order


Ping-Pong Type – intermediate Ps are released even before substrates are added

Regulation of Enzyme Activity

- Allosteric interactions –allosteric sites bind with (+) or (-) effectors at activator or inhibitory sites, alters enzyme conformation, K class (alters Km) or V class (alters Vmax), sigmoidal curve of v vs [S],

o oligomeric allosteric enzymes – several subunits, indentical = protomers, ligands may affect binding of ligand to other protomers

" Homotropic – affects same ligand to protomer

" Heterotropic – affects different ligand

- Reversible Covalent Modification – covalent attachment of small groups affects catalytic properties, hydrolyze to reverse

- Stimulation and Inhibition of Control Proteins – active when Ca

- Proteolytic activation – inactive precursors (zymogens or proenzymes) converted irreversible to active forms by hydrolysis of some peptide bonds

Applications in Clinical Diagnosis

- intracellular enzymes released into plasma upon cell damage and death

- isoenzymes – same enzyme, different physical and chemical properties, located in different places

- therapeutic agents

- immobilized enzymes as reagents in diagnosis kits

- indicators for ELISA – antibody detection

therapeutic agents - immobilized enzymes as reagents in diagnosis kits - indicators for ELISA – antibody