ENZYMES

- biological catalysts, increase the rates of chemical rxn by lowering Ea (energy reqd to produce
transition state), net energy 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
o Class 1 Oxidoreductases redux rxn
o Class 2 Transferases transfer chemical groups
o Class 3 Hydrolases bond cleaving with water
o Class 4 Lyases nonhydrolytic splitting of molecules or addition of groups to DBs
o Class 5 Isomerases interconvert isomeric molecules
o 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 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
o Competitive resembles substrate, competes for active site, overcome by substrate conc
o Non-competitive binds to allosteric site, deactivating enzyme before binding
o Uncompetitive acts in ES complex, alters turnover rate

Enzyme Kinetics
- Units Specific activity (mol/min per mg protein), Turnover Number or Catalytic constant (Kcat,
mol/min per mol enzyme), Katal (kat, amount of enzyme acitivty that transforms 1 mole of substrate
per second)
- Assumptions of Michaelis and Menten
o Enyzme reacts reversible, forms ES complex
o [S]>>[E], possible to saturate E as ES with excess S
o 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
o Total enzyme conc Et = free enzyme E + bound enzyme ES
o Michaelis Menten Equation: V = Vmax [S] / Km + [S]
- Significance of Km and Vmax
o Km = [S] at Vmax
o measures affinity of an enzyme to substrate: Km = weak binding, Km = strong binding
o Vmax reveals turnover number (Vmax = k3[Et])
- Graphing
o Michaelis Menten Graph rectangular hyperbola, Vmax, Vmax, km
o Lineweaver Burk Equation reciprocal of MME, linear slope, y intercept = 1/Vmax, x = 1/Km,
slope = Km/Vmax
o Eadie Hofstee plot v=-Km x v/[S] + Vmax, slope = -Km
- Effects of Inhibition on LBE graph:
Inhibition
Vmax
Km
Competitive

Noncompetitive

Uncompetitive

- Multisubstrate Kinetics
o > 1 substrate interactions may be bi-uni (2S, 1P) or bi-bi (2S, 2P)
o Cleland notation system illustrates rxn categories
o 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
o 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