Transcript Slide 1

Enzymes
•
They control metabolism by regulating metabolic reaction rates:
molecules that accelerate or catalyze chemical reactions in cells
by breaking old covalent bonds & forming new covalent bonds
•
Except for Ribozymes, all enzymes are proteins
•
a biological catalyst…
• have complex structure (sequence of aa’s)
• act only upon a specific substrate (or substrate group)
• do not change the energetics of the reaction
Enzyme Action
E + S <---> [ES] <---> E + P
enzymes catalyze reactions by lowering the energy of activation (Ea)
What does an ES Complex do?
-
holds substrate out of aqueous solution
holds substrate in specific orientation, close to Transition State to
allow reaction to occur
reduces ability of free rotation & molecular collisions with nonreactive atoms
allows an altered local environment: changes ionic strength, pH,
adds or removes H-bonds to substrate
Historical Background
2100 BC
Codex of Hammurabi-description of wine making
700 BC
Homer’s Iliad:
As the juice of fig tree curdles milk, and thickens it in a moment
though it be liquid, even so instantly did Paeeon cure fierce Mars
1700s
Late 1800s
1903
1913
1950s-1960s
1965
Réaumur - studies on the digestion of buzzardsdigestion is a chemical rather than a physical process
Kühne - term 'enzyme': Greek "in yeast"
Hans & Eduard Buchner – filtrates of yeast extracts
could catalyse fermentation! No need to living cells
E. Fischer – “lock and key” hypothesis
Henri – first successful mathematical model
Michaelis and Menten – NZ rate equation....
Koshland – “Induced fit” model
Monod, Wyman and Changeux – allosteric regulation
Terminology
Many enzymes require a non-protein component for activity:
• cofactor: small inorganic ions... mostly metal ions: Cu (cytochrome
oxidase), Mg (kinases), Fe (catalase, peroxidase)
• coenzymes: small non-protein but organic compounds
Coenzyme A: acyl transfer
Flavins: redox reaction
NAD+ (NADP+): redox reactions
Vitamins: derivatives of B vitamins (B1, B2, B6, B12), niacin, folic
acid, riboflavin
• prosthetic group: tightly bound large complex organic molecules,
(heme)
Holoenzyme vs apoenzyme (apoprotein)
• active site: portion of enzyme which folds to precisely fit the contours of
a substrate via weak electrostatic interactions & facilitates bond
reactivity
• allosteric site: a site other than the active site
Isoenzymes
• Classification is based on reaction catalyzed so enzymes isolated
from different organisms but catalysing same rxn have same
number but different amino acid sequence
• Even within a single species, there may exist different forms of
enzyme catalysing the same reaction. Differences may be:
– A.acid sequence
– Some covalent modification
– 3-D structure
• Isoenzyme (isozyme): different variants of the same enzyme having
identical functions
Properties of enzymes as catalysts-1
Catalytic power
• They may increase reaction rate by as much as 1015-fold
2H2O2
2H2O + O2
No catalyst
Fe2+ catalyst
Catalase
Rate (L/mol/s)
1 x 10-7
56
4 x 107
Specificity
• Most enzymes are highly specific to their substrate and reaction
catalysed
– Bond specificity: e.g peptidase, phosphatase
– Group specificity: e.g hexokinase
– Absolute or near-absolute specificity
• Stereospecificity:
– Dehydrogenases catalyst the transfer of hydrogen from the substrate to
a particular side of nicotinamide ring in NAD+ or NADP+
– Phenylalanine hydroxylase uses L-Phe not D-Phe
• Importance of specificity in DNA replication and protein synthesis
 proofreading
Properties of enzymes as catalysts-2
Regulation
• Allosteric regulation (+/- effectors)
e.g. feedback inhibition
• Covalent regulation (phosphorylation by ATP-dependent protein
kinases)
e.g. Glycogen phosphorylase
• Activation of zymogens, which are inactive proenzymes
e.g. trypsinogen
• Amount of enzyme:
– gene expression
– enzyme degradation
How to define enzyme activity?
Physical properties of an enzyme most often is measured by relative
rate that substrate ---> product
• 1 unit ACTIVITY= International unit (IU)
amount enzyme which converts 1 μmole substrate per min at 25oC
– e.g. IU= 10 μmole/min
• 1 unit SPECIFIC ACTIVITY
# IU of enzymatic activity per mg of total protein present
– e.g. 10 μmole/min/mg protein or 10 IU/mg protein
Classification of Enzymes
Enzyme Commission (EC, 1955) - IUBMB International Union of
Biochemistry & Molecular Biology
4 digit Numbering System
1st
2nd
3rd
4th
[1.2.3.4]
one of the 6 major classes of enzyme activity
the subclass (type of substrate or bond cleaved)
the sub-subclass (group acted upon, cofactor required, etc...)
a serial number… (order in which enzyme was added to list)
Major Classes of Enzymes-1
1. Oxidoreductases [dehydrogenases, oxidases, peroxidases]
oxidation-reduction reactions, often using coenzyme as NAD+/FAD
Alcohol dehydrogenase [EC 1.1.1.1]
CH3CH2OH + NAD+ ---> CH3CHO + NADH + H+
2. Transferases [kinase, phosphorylase, transaminases]
group transfer reactions (AX + B
BX + A)
Hexokinase [EC 2.7.1.2]
D-glu + ATP ---> D-glu-6-P + ADP
3. Hydrolases [digestive enzymes; amylases, lactase, sucrase]
hydrolytic reactions: (AX + H2O
XOH + HA)
Alkaline phosphatase [EC 3.1.3.1]
R-PO4 + H2O ---> R-OH + H-PO4
Major Classes of Enzymes-2
4. Lyases [decarboxylases]
elimination rxns in which a double bond is formed
Pyruvate decarboxylase [EC 4.1.1.1]
pyruvate ---> acetaldehyde + CO2
5. Isomerases [mutases, cis-trans isomerases, racemases]
isomerization rxns
Alanine racemase [EC 5.1.1.1]
L-alanine ---> D-alanine
6. Ligases [a.acid RNA ligase]
condensation of 2 substrates at the expense of energy (ATP)
(X + Y + ATP
XY + ADP + Pi)
Isoleucine-tRNA ligase [EC 6.1.1.5]
L-isoleucine + tRNAIle + ATP ---> L-isoleucyl- tRNAIle + ADP + PPi
Multienzyme systems
• Proteins that exhibit more than one catalytic activity
• EC recommendation  more than one catalytic activity  system
e.g. fatty acid synthase system
• Multifunctional enzymes will have more than one EC number...
• Multifunctional enzyme can made up of:
– Several polypeptide chains with different catalytic activities may be
associated with each other
– A single polypeptide chain with multiple catalytic site
– or even both
Tools of enzymology-1
Spectroscopic techniques (structure and reactivity in solution)
• Optical (circular dichroism, UV-visible, fluorescence)
• Vibrational (infrared, Raman)
Electrochemical methods (kinetic analysis)
• Potentiometric techniques
• Conductometry
Enthalpimetry (microcalorimetry)
• Very sensitive and free of interference
Radiochemical methods
• Far more sensitive than photometric ones but...
Tools of enzymology-2
X-ray crystallography
• First crystallized enzyme, urease (J. Sumner, in 1926)  crystals
are proteins and their dissolution led to enzymatic activity
• Within 20 years: >130 enzymes crystals documented
• 3-D structure of a protein, myoglobin, was deduced (Kendrew, 1957)
Multidimentional nuclear magnetic resonance (NMR) and X-ray
crystallography are now commonly used:
– to explain the mechanistic details of enzyme catalysis
– to design new ligands
Molecular Biology
• Clone and express enzymes in foreign hosts (overexpression 
purification and characterization of enzymes occuring naturally in
minute quantity)
• Manipulate the a.acid sequence (site-directed mutagenesis and
deletional mutagenesis  chemical groups in ligand binding)