COENZYMES

- heat stable, low MW, organic, accept and transfer f(x)al groups, non-protein (cofactor/prosthetic group) of holoenzyme
Coenzyme
Classification /
Group Transferred
Components Active site
Binds to
apoenzyme
Vitamin
derivative
Utilized by
NAD+ / NADP + Redox
AMP (nucleotide), nicotinamide
(pseudonucleotide -pyridine derivative),
quinonoid when reduced
C4 of pyridine ring
Via adenine, ribose &
pyrophoshate groups
Niacin/nicotin
ic acid
LDH, MDH, reductive biosynthesis of
fats, G-6-P rxn
FAD / FMN Redox
Isoalloxazine ring, ribitol (sugar alcohol of
ribose), semiquinone radical when
reducing
N1 & N5 of
isoalloxazine ring

Riboflavin
(B2)
Acyl-CoA DH, L-amino acid oxidase,
oxidation of succinic to fumaric acid
(succinate DH)
Coenzyme Q Redox
Benzoquinone ring with repeating
isoprenoid units
C1 & C4 of
benzoquinone ring
N/A
NADH-CoQ DH (complex III of
respiratory chain)
Tetrahydro-
biopterin
Redox
N/A, 2x
reduced
biopterin
Hydroxylation of aromatic AA (P, W)
Thiamine
pyrophosphate
(TPP)
Acyl and active
aldehyde, ketol
Substituted pyrimidine linked to thiazole
ring w/ terminal pyrophosphate,
inactivated by thiaminase (raw fish),
requires Mg++
C2 of thiazole ring
Thiamine
(B1)
Oxidative (PDH) & non-oxidative (PDC)
decarboxylation of pyruvic acid,
transketolation
Lipoic acid /
thioctic acid
Acyl and active
aldehyde, acyl and
H+
Exists as cyclic (oxidized) or open chain
(reduced, dihydrolipoic acid)

amino group of
lysine as amino-
lipoyl-L-lysine
N/A
Oxidative decarboxylation of -keto
acids, cofactor of pyruvate DH &
ketoglutaric acid DH
Coenzyme A
Acyl and active
aldehyde, forms
thioacyl derivatives
AMP, pyrophosphate, pantothenic acid,
thioethanolamine
Nucleophilic SH of
CoASH

Pantothenic
Acid
Fatty acyl CoASH synthase,
dihydrolipoyl transacylase of PDH
Biocytin
Activation and
transfer of CO
2

Imidazolone ring fused with
tetrahydrothiophene linked to valeric acid,
synthesis inhibited by avidin (raw egg
white)
N of imidazolone
ring
amino group of
lysine
Biotin
Carboxylation or carboxyl transfer,
biotin-dependent carboxylases (Acetyl-
CoA, pyruvate, propionyl &
methylmalonyl CoA), carboxylation of
substrates by accepting ATP-activated
carboxyl group & transferring it to
carboxyl acceptor
Pyridoxal
phosphate
Amino group
Exists as pyridoxal PO4 or pyridoxamine
PO4, inhibited by isoniazid (INH), reacts
with -amino group = Schiffs base
Aldehyde group of
C4 of pyridine ring

Pyridoxine
(Vit B6)
Transamination , decarboxylation
(AADC), racemization (DL) (AA
racemase)
Tetrahydrofolatic
acid
One C group
(methyl, methylene,
methenyl, formyl,
formimino)
Substituted pyridine ring linked to p-
aminobenzoic acid (PABA) bound to
glutamic acid, inhibited by methotetraxate
& sulfonamides (folate antagonists)
N5 of pteridine ring
& N10 of PABA for
transfer of 1-C
groups

Folic acid
(successive
reductions)
Serine hydromethyl transferase,
thymidylate synthetase, formyl
transferase
Cobamide
coenzyme
Alkyl group
Tetrapyrrole ring with central cobalt atom,
exists as deoxyadenosyl cobalamin or
methylcobalamin

Cyano-
cobalamin
(B12)
Methylmalonyl CoA mutase,
(methylmalonyl succinyl)
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