Chemistry: Enzyme Mechanism and Regulation
Active sites of Enzymes
What are Enzymes?
biological catalysts (speed up biochemical
reactions)
 Most are proteins composed of one or more
polypeptide chains
 Operate under mild conditions:
 room/body temp
 pH near 8
 aqueous solution
 modest amount of salts present
Substrates – the compounds acted on by enzymes
Catalytic power – acceleration of the reaction over that
for the noncatalyzed reaction
Substrate specificity – enzymes action on a limited set of
substrate. Only one or a few chemically related
compounds.
ex. Amylase: starch
Lipase: fats (digestive enzymes)
I. Cofactors
Not all types of reactions can be catalyzed by amino
side chains alone. Enzymes often use small
organic/inorganic molecules for assistance in
performing these other type of reactions(cofactors)
I. Cofactors
2 main subcategories of enzyme cofactors:
1. Prosthetic groups – tightly bound
cofactor(often covalently bond) ex. Flavins,
heme groups, biotin
2. Coenzymes – sometimes called cosubstrate. Is
distinguished by it’s relative freedom. Held only
loosely by the enzyme, and can be released and
taken up again relatively freely.
Holoprotein/holoenzyme – the complete complex of a
protein with all necessary small organic molecules,
metal ions & other components
Apoprotein/apoenzyme – the remaining protein when
the non-proteinaceous part is stripped away
ex. Myoglobin; x heme group  apomyoglobin
II.
- Is the region that binds substrates, cofactors,
prosthetic groups and so on.
- Contains the residues that help hold(bind) the
substrate or that directly participate in the
reaction
- a three dimensional cleft or crevice formed by
groups that come from different parts of the
amino acid sequence
- Precise folding aligns functional groups in the
active site for specificity in binding substrate &
in the catalyzing the reaction
The microenvironment around the site may change the
properties of functional groups. (e.g., local electric
fields, polarizability of the sorroundings, solvent
accessibility)
Only a few residues in the active site participate directly
in catalytic action:
• Polar residues – participate in hyodrogen-
bonding & electrostatic interactions and short-
range van der Waals interactions.
• Nonpolar side chain residues – may help in
binding and aligning the substrate & cofactor/s
& can help[ enzyme recognition & acceptance
or rejection of potential substrate moilecules,
just on the basis of molecular shape
III. Conformational change
- Proteins are flexible.
- Binding of substrate, inhibitor or other effectors
of activity can cause either small or large
conformational changes in an enzyme
A. Lock-and-key model
B. Induced-Fit model
A. Lock-and-key model
⁻ Substrate(Key) must fit into the active
site(lock) of the enzyme precisely
⁻ Often interpreted to
mean that the enzyme
does not need to change
shape or conformation
to catalyze the reaction.
B. Induced-Fit model ⁻ The pH range over which a particular side chain
can do this will strongly depend on the chemical
⁻ The functional groups may have to be moved nature of that side chain’s ionizable group.
into position for correct alignment
 Nucleophilic catalysis
⁻ there is not necessarily a good steric fit initially. ⁻ using covalent bonds between enzyme and
substrate, with an amino side chain on the
⁻ real enzymes tend to use an induced-fit enzyme acting as a nucleophile
mechanism. Enzymes are rarely rigid. ⁻ nucleophile (nucleus-loving) : they attack
⁻ The shape change electron-poor regions on the substrate
might call for conformational ⁻ Ex. Serine, threonine, tyrosine (hydroxyl
changes elsewhere in the groups)
protein as well.  Electrophilic catalysis
⁻ The enzyme withdraws electron density from a
⁻ Also, there may be a distortion of the reaction center
conformation of the substrate as it fits into the  Proximity effects
active site which could aid in catalysis. ⁻ Aligning and juxtaposing(put close together)
moieties on substrate and enzyme for rapid
IV. Enzymatic Catalysis
reaction
Molecularity of a reaction – number of ⁻ Important for enzymes acting on two
particles(molecules, ions, atoms) altered in a reaction substrate molecules or substrate and
cofactor molecules simultaneously.
AP:unimolecular  Bond strain
A + B  P : bimolecular ⁻ An enzyme may change its conformation to
Transition state cause the substrate’s bonds to approximate
- highest point of the barrier ΔG++ those of the transition state. This generally
- very brief weakens the bond and makes it more reactive.
 Charge neutralization
Enzymes basically achieve their catalytic power through ⁻ This can facilitate substrate binding or help
a reduction in the height of the barrier ΔG++ stabilize an intermediate
Enzymatic binding to the transition state and rate  Environmental effects
acceleration ⁻ Here the solvent or other molecules help to
stabilize the transition state thereby promoting
Uncatalyzed Enzyme-catalyzed reaction
reaction reaction VI. Classes of Enzymes and Types of Reactions

height of the HIGHER lower

barrier : ΔG++
Activation HIGHER lower
energy
rate slower FASTER

V. General Mechanisms for Catalysis

 General acid-base analysis
⁻ Using a moiety (usually an amino acid side • Suffix: -ase
chain) in the enzyme (odler names may not reflect this convention
⁻ The side chain must be capable of donating or ex. Trypsin, Pepsin)
accepting protons and it should do so reversibly
to regenerate the original state of titration of • Abbreviations:
the enzyme. Ribonuclease  Rnase
 • Under modern conventions, name of an enzyme
is constructed to indicate both the type of
reaction and the principal substrate or product
of the reaction
Ex. Dehydrogenase  reductase & oxidase :
redox reaction
RIBONUCLEASE A: Acid-Base Catalysis
•Rnase A is a digestive enzyme that hydrolyzes RNA.
•The enzyme forms a 2`,3` cyclic phosphate
intermediate in a two staged mechanism.
•There is a strong pH dependence, with a characteristic
of a bell shaoe activity.
SUBSTRATE BINDING
•The binding site contains several proton/acceptor
groups-His 12, His 119, Lys 7, Lys 41, and Lys 66.
•There is a high specifity for the pyrimidine ring on the
3` side of the cleaved bond.
•There is a low specifity for any particular base on the
5` side, binding here is through ionic attraction.
MECHANISM
The reaction can be divided into two stages, with each
of its own transition state, going to and from semi-
stable intermediate.
FIRST STAGE
1.) Unprotonated His 12 takes a proton from the 2`-OH,
while protonated His 119 starts to donate proton to the
5-O. Simutaneously the 2-O starts to bond to
phosphorus, forming a pent covalent intermediate.
2.) The bond between P and the 5`-O breaks and the
proton is finally transferred from His 119 to the oxygen.
The bond between P and 2`-O is fully formed.
Second stage
3.) The 5-OH end leaves the site and molecule of water
enters.
4.)Ionized His 12 donates a proton to 2`,3` cyclic
phosphate as neutral His 119 takes a proton from H2O
leaving OH next to cyclic phosphate.
5.)OH attacks the cyclic phosphate, forming a pent
covalent intermediate.
6.)The 2-OH is reformed, with the 3` carbon carrying the
phosphate group.
Chymotrypsin and other Proteases
•Chymotrypsin is a protease; it is a digestive enzyme,
secreted by the pancreas. It cuts the amide backbone of
proteins and prepared segregation to amino acids and
peptides.
•Chymotrypsin cleaves peptides on the carboxyl side of
aromatic or large nonpolar residues
•The amide linkage of the substrate contains three
residues: Asp 102, His 57, and Ser 195-they form
catalytic triad that facilitates the cleavage.
•Chymotrypsin and its relative that uses the same
reaction mechanism are known as serine proteases.
REACTION MECHANISM FOR CHYMOTRYPSIN
First stage
1.) A substrate peptide chain enters the active site. Asp
102 is already ionized and forms a strong hydrogen
bond on the ring of His 57
2.) The Ser 195 anion attack the peptide carbonyl,
forming a tetrahdral transition state.
3.) The transition state quickly collapse and leaves the
substrate carbonyl attached to the side chain of Ser
195.
4.) The remainder of substrate chain leaves the active
site.
Second Stage:
5.)Water enters the active site, His 57 became
polarized.
6.) The polarized water attacks the acyl-enzyme
carbonyl and forms another tetrahedral transition state.
7.) The tetrahedral transition state collapse with part of
the water.
8.) The carboxylic acid leaves the active site, which has
now been regenerated and ready to accept another
substrate molecule.
CARBONIC ANHYDRASE: Metal Ions and Electronic
Strain
Carbon dioxide (𝒄𝒐𝟐 )
-is produced by the cell in aerobic metabolism of
carbohydrates, lipids and amino acids.
-𝑐𝑜2 rapidly and spontaneously reacts with water to
produce the relatively strong acid, carbonic acid, which
then spontaneously ionizes to release a proton and
generate bicarbonate ion.
Carbonic Anhydrases (CA)
-are enzymes that performs the conversion of carbon
dioxide to bicarbonate in the red blood cells, speeding
up the naturally occurring reaction.
Reaction Mechanism
Steps in Reaction
Step 1: Water loses proton generating a Zn bound
hydroxide ion.
Step 2: Carbon dioxide binds the active site which
positions perfectly to interact with the hydroxide ions.
Step 3: The ions attacks 𝑐𝑜2 converting it into a
bicarbonate ion.
Step 4: A second molecule of water enters the active
site displacing bicarbonate and starting the process over
again.
Allosterism
-is defined as the situation where activity of a protein is
altered as a consequence of some molecule binding at a
site different from the active site. (allosteric behaviour)
Cooperativity
is a phenomenon displayed by systems
involving identical or near-identical elements, which act
dependently of each other, relative to a hypothetical
standard non-interacting system in which the individual
elements are acting independently.
THE CONCERTED or MWC MODEL OF ENZYME
ALLOSTERISM AND COOPERATIVITY
Monod, Wyman and Changeux (MWC) were the authors
of the paper that first described the concerted model.
BASIC ASSUMPTIONS IN THE MWC Model
• The enzyme has two or more identical subunits
• Subunits can switch between two (or more)
conformations--- ex. R and T
• Each subunit has one or more binding sites
• The affinity of the site for the ligand depends on
the subunit’s conformation; the R conformation
is usually chosen to indicate the one with higher
affinity (or activity) for ligand.
THE SEQUENTIAL OR KNF MODEL
KNF (Koshland, Nemethy, and Filmer) the author of the
paper that first proposed the sequential model.
BASIC ASSUMPTIONS
• The KNF model applies to multisubunit
enzymes, whose subunit switch between two or
more conformations. There are possibly several
binding sites per subunit, whose affinity or
activity depends on the conformation of the
subunit.
• The KNF model can change conformation one at
a time.
STRUCTURE AND CONFORMATIONAL CHANGES
The enzyme shows large conformational
changes and there is a high degree of cooperatively
present, making this system a good example or
allosterism and cooperatively.
Enzyme has multiple subunits of 2 different types:
•1st subunit type - denoted as type r. -regulates the
activity but does not catalyze the reaction -this subunit
type binds nucleotides like ATP or CTP, then alters their
conformation to affect the activity occuring at the
second type of subunit.
•2nd subunit type- denoted as type c. -where catalysis
takes place -it binds the substrates of enzyme,
aspartate, and carbamoyl phosphate.
Upon binding substrate, large changes occur.
CATALYTIC MECHANISM
Two substrates are available to bind into the catalytic
site: carbamoyl phosphate and aspartate.
There is a well defined temporal order to binding, first- 2. Glycosylation - with attachment of sugar or
carbamoyl phosphate then aspartate. carbohydrate, for targeting and molecular recognition.
 Carbamoyl phosphate -is relatively unstable in 3. Methylation and Acetylation
solution (it has a half life of approximately 5
minutes at 37°C) so, it makes sense to bind it
and hold onto it to protect it from the
degradation in free solution. Later when
aspartate becomes available, carbamoyl
phosphate is rapidly converted to product.
REGULATION BY COVALENT MODIFICATION
 The amplification of the original
bonding(hormone bonding) is achieved by using
signaling cascades that rely in a part on a series
of covalent modifications of a set of enzymes in
the cell.
 hundreds or thousands - substrate molecules,
the binding of one hormone molecule leads to
many thousands of chemical changes inside the
cell.
 cascades use covalent modification such as
phosphorylation or peptide cleavage to achieve
this signal amplification.
REVERSIBLE PHOSPHORYLATION
 The modification here is the attachment or
removal of phosphoryl groups on the side
chains of certain amino acids.
 The cofactor ATP is typically the donor of the
phosphoryl group.
 Attachment of a phosphoryl group is performed
by enzymes called protein kinases.
 Removal of the group is performed by protein
phosphates. The use of ATP provides a link to
the overall state of the cell in terms of its stores
of energy.
PROTEOLYSIS

 Process by which such digestive enzymes as

trypsin and chymotrypsin are formed;
proteolytic processing of their catalytically inert
precursors, trypsinogen and chymotrypsinogen,
is needed to generate the catalytically
competent enzymes.

OTHER MAJOR TYPES OF COVALENT MODIFICATION

Several other types od covalent modifications have

been found. Some common changes are profiled here:

1. Acylation - involving lipid attachment, for targeting

the protein to membranes.