Module 1

Hydrogen bond is a weak non-covalent interaction between the hydrogen attached to an

electronegative atom in one molecule and an electronegative atom of a second molecule.
Organic compounds that form hydrogen bonds with water are usually soluble in water.
Ion Product of water: Kw = [H+][OH-] = 1 x 10-14
In other words, the ion product of water is equal to concentration of hydrogen ion times the
concentration of hydroxide ion. The brackets stand for concentration so, if H+ stands for hydrogen
ion, [H+] stands for the concentration of hydrogen ion. The units are moles per liter.
If [H+] increases, then [OH-] decreases and vice versa.
At pH = 7.0, the solution is neutral and both the [H+]and the [OH-]are 1 X 10-7, that is Kw = [H+][OH-]
= [1 X 10-7][1 X 10-7] = 1 x 10-14
If a solution has more [H+] than [OH-], the solution is acidic. If a solution has more [OH-] than [H+], the
solution is basic.
To solve this type of problem, the equation can be manipulated to solve for either the [H+] or the [OH-]
as shown below:
Kw/[H+]=[OH-] or Kw/[OH-]=[H+]
Example: If the concentration of H+ is 1x10-6, what is the concentration of OH-?
By using Kw/[H+]=[OH-]:
1x10-14/1x10-6 =[OH-]
[OH-] = 1x10-8
pH is the negative log of the concentration of hydrogen ions (such concentration expressed in moles
per liter [mol/L]) in a solution. This determines the acidity of the solution.
In Mathematical terms, pH = -log[H+], where [H+] is hydrogen ion (or proton)
Acids and Bases
Acid: An acid is a proton donor, i.e., any chemical species (molecule or ion) that is able to '''lose or
"donate'''" a hydrogen ion (proton) in solution.
When dissolved in water, an acid creates a solution with a pH lower than 7.0.
Base: A proton acceptor, i.e., any chemical species (molecule or ion) that is able to ''gain or "accept"
a hydrogen ion (proton) in solution.
When dissolved in water, a base creates a solution with a pH higher than 7.0.
Conjugate base: is the ion or molecule remaining after the acid has lost a proton and is also referred
to as the salt of the acid.
Strong Acids:
Strong acids are completely dissociated at any pH.

When added to aqueous solution, strong acids completely dissociate into the salt of the acid
and protons
Besides sulfuric acid (H2SO4), the body also produces hydorchloric acid (HCl) and nitric acid
(HNO3)
Strong acids cannot form buffers.

Weak Acids:
Weak acids only partially dissociates into the salt of the acid (conjugate base) and protons
when added to water. The ratio of salt to weak acid can then be adjusted by adding H+ or OHto the solution.
Only weak acids can buffer aqueous solutions.
'The equation for its dissociation of a weak acid: HA = H+ + AHA: weak acid concentration,
A- : conjugate base concentration
H+ :proton concentration
Definition of the dissociation constant (Ka): Ka=[H+][A-]/[HA].
Ka = dissociation constant
[HA]= weak acid concentration
[A-]= conjugate base concentration
[H+]= proton concentration
Relationship between pKa and Ka: pKa=-log(Ka)
Henderson-Hasselbalch equation: pH = pKa + log ([A-]/[HA])
When the [A-] = [HA], the pH = pKa

Increasing the [HA](acid) brings the pH down (Inverse relationship)

Increasing the [A-](base) brings the pH up (Direct relationship).

Buffers:
Two factors that determine effectiveness of the buffer:
1. The pKa of the buffer relative to the pH of the solution.
2. The concentration of buffer present (must have enough buffer molecules per unit volume to
recombine or dissociate with hydrogen ions).
Buffers are most effective:
When the pH of the solution is equal to the buffer's pKa.
Generally, when the pH of the solution is within one pH unit of the buffer's pKa (pH = pKa +/-1).
Explanation:
When an acid (H+) is added to a buffered solution, the salt of the buffer, (the conjugate base),
combines with the added acid and prevents the pH from changing as much as it would have without
the buffer present.

When a base (OH-) is added to a buffered solution, the acid of the buffer combines with the added
base and prevents the pH from changing as much as it would have without the buffer present. (See
figure 4.7, Marks Medical Biochemistry)
Physiological pH
A physiological pH is necessary to maintain protein structure and retain function.
Proteins, have a narrow range of pH in which they function correctly. Changing the pH changes the
structure of the proteins and this, in turn, changes their functions. Too much change may result in the
failure of enzymes and biological pathways.
Protein structure and function:
Primary Structure:
Is linear sequence of amino acids in polypeptide chain. Written from the amino terminus (N-terminus)
to the carboxyl terminus (C-terminus).
Secondary Structure:
Alpha helix: Alpha helices are formed when carbonyl group of peptide bond forms a hydrogen
bond with the amide nitrogen of another peptide bond four amino acids down the polypeptide
chain.
o The peptide backbone is formed by hydrogen bonds between each carbonyl oxygen
atom and the amide hydrogen located 4 residues down the chain. This unique bonding
sequence results in an alpha helix structure that is highly compact and rigid.
o Comprises about a third of all secondary structures.
o R-groups protrude from the helix
o See Figures 7.3 and 7.4 Marks Medical Biochemistry

-pleated sheets: -sheets are formed when beta strands are connected laterally by at least
two or three backbone hydrogen bonds.
o Are made up of Beta strands that are almost fully extended in the sheet.
o The beta strands are connected horizontally by hydrogen bonds formed between C=O
groups of either strand and NH groups of either strand.
o pleated sheets can either be parallel or anti-parallel.
o The R groups always protrude to the top or bottom.
o Depending on the R groups on the sides of the beta sheet, the sheet may have a
hydrophilic and a hydrophobic side.. The hydrophilic side of the sheet will be on the
surface of the protein and be exposed to the polar H2O solvent. The hydrophobic side of
the sheet will be buried in the protein and will only rarely be exposed to the polar H2O
solvent.
o See Figure 7.5 in Marks Medical Biochemistry

bends, turns, or loops: Short stretches areas of the polypeptide chain form these structures
that are stabilized by hydrogen bonds (these are not random)

Tertiary Structure:
Tertiary structure is the total 3-D conformation of an entire polypeptide chain including interactions
between alpha-helices, beta-sheets and any other loops, turns, or bends. (See figure 7.8, Marks
Medical Biochemistry)
Actin fold is an example of tertiary structure.
Structural domains have specific function
Motifs are common arrangements of secondary structures to generate a tertiary arrangement

Quaternary Structure:
A combination of two or more tertiary subunits that work together as one functioning unit.
Apoprotein: A protein missing its ligand or ligands. Example: hemoglobin lacking heme
Holoprotein: A protein with its ligand so it is able to function. Example: hemoglobin bound to heme
Apoenzyme: An enzyme missing its cofactor or cofactors. When combined with the proper cofactors,
usually a metal ion or a coenzyme, the apoprotein becomes an active enzyme (holoenzyme).
Myoglobin and Hemoglobin:
Both bind oxygen
Hemoglobin travels in the blood inside a red blood cell to deliver oxygen to tissues.
Myoglobin remains in the heart and skeletal muscle cells to bind oxygen released by
hemoglobin.
Homologous proteins; they have similar primary sequences
Like all proteins, the tertiary and quaternary structures are stabilized by hydrophobic
interaction, hydrogen bonds, and salt bonds
Myoglobin:
is a monomer has 8 alpha-helices linked together by alpha-turns
has a hydrophobic pocket containing heme with an ferrous iron atom (Fe+2) at its center for
oxygen binding.
Fe2+ is always bound to a histidine R-group of the alpha helix. This binding stabilizes the
reduced state of iron when it binds to oxygen
heme is tightly bound to the globin, it is termed a prosthetic group.
Myoglobin binding hyperbolic
Hemoglobin (Hb):
Hb is a heterotetramer (four "mers" and at least one monomer is different from the others).
Hb is composed of 2 alpha and 2 beta subunits.
Each subunit has its own heme; hemoglobin can bind 4 oxygen molecules
Cooperativity: When O2 binds to the Fe2+ at one of the Hb binding sites, it pulls on the
histidine, which pulls on the alpha helix, changing the conformation of the globin slightly. This
slight movement changes the conformation of the other three chains in the Hb.
When oxygen binds to one heme, the other hemes are more likely to bind a second molecule
of oxygen.
Hemoglobin does not by oxygen as strongly as myglobin sigmoidal binding
When oxygen is released from hemoglobin, the loss of one molecule of oxygen facilitates the
loss of additional oxygen molecules, This is the reverse of positive cooperativity'.
Hemoglobin can exist in two conformations: the T-state or the R-state.
In the T (tense) state, Hb has a low affinity for O2.
In the R (relaxed) state, Hb has a high affinity for O2.
Bohr effect: impact of pH on oxygen binding hemoglobin. A decrease in pH decreases
hemoglobin satruation
HbA1c is made spontaneously in the blood from hemoglobin and glucose. A molecule of glucose is
bound nonenzymatically and irreversibly to the amino terminus of a beta chain of hemoglobin. Since
the concentration of hemoglobin and the temperature of the blood are constant, the percent of HbA1c
depends only upon the concentration of glucose in the blood.

HbA1c measures the average glucose in the blood for the past 6-8 weeks.
An HbA1c level > 5.2% is considered pathological.
Enzymes as catalysts
Enzymes:
The substrate specificity of an enzyme is the ability of an enzyme to select one or a few
substrates from a group of similar substrates.
The active site contains functional groups that participate in the reaction.
The reaction takes place away from water solution.
The enzyme usually changes conformation due to the interactions between the amino acid
side chain groups of the enzyme and the functional groups of the substrate, so that the outside
solution can't take part in the reaction
Enzymes increase the rate of the reaction by decreasing the activation energy, i.e., stabilizing
the transition state.
Lock and key model: It was originally thought that a rigid substrate would slide into a rigid active site
of the enzyme and a reaction would take place (as a key fits into its matching lock and then works to
open the lock).
Induced fit model: The enzyme changes its conformation when it binds to a substrate and this
"induced" conformation is due to the interactions between the amino acid side chains of the active
site and the functional groups of the substrate; the substrate also changes conformation in response
to the enzyme.
Transition State: State during an enzyme reaction when an intermediate that resembles both
substrate and product, and contains the most free energy, exists. The enzyme stabilizes the transition
state by lowering its activation energy.
Activation Energy: Energy necessary to achieve the transition state. Since the rate at which a
substrate can become product depends on the rate at which the enzyme/substrate complex can
reach the transition state, the activation energy determines how fast a reaction will go.
Cofactors:
Three general categories of cofactors are: coenzymes, metal ions, and metallocoenzymes (similar to
heme in hemoglobin).
Tightly bound cofactors are termed prosthetic groups.

Coenzyme: is any organic cofactor that binds to the enzyme and is necessary for the reaction.
They are usually inert when not bound to their respective enzyme

Reactions:
Acid- base catalysis activates the substrate by interaction with an acidic or basic amino acid R
group to initiate a reaction
o Example: chymotropsin (See Figure 8.9 Marks Medical Biochemistry)
A Covalent catalysis activates the substrate for transfer by forming a covalent bond with a
portion of the substrate that contains a lot of free energy so that the transfer of the group is
exergonic.

o Example: TPP (See figure 8.11 in Marks Medical Biochemistry)

Types of activation transfer coenzymes:

o Thiamine pyrophosphate: synthesized from Thiamine, B1)
o Coenzyme A (CoA) (pantothenic acid): is synthesized from the vitamin pantothenic acid
(or pantothenate, B5)
o Biotin: a water soluble B-complex vitamin, B7
Carboxylase Enzymes use biotin.
o Pyridoxal phosphate (also called B6)
Pyridoxal phosphate usually reacts with the amino group of an amino acid.

General classes of enzymes:

1. Oxidoreductase: Oxidoreductases catalyze oxidation reduction reactions. At least one
substrate becomes oxidized and at least one substrate becomes reduced.
2. Transferase: Transferases catalyze group transfer reactions- the transfer of a functional group
from one molecule to another.
3. Hydrolase: In hydrolysis reactions, C-O, C-N, and C-S bonds are cleaved by addition of H2O
in the form of OH- and H+ to the atoms forming the bond.
4. Lyase: Lyases cleave C-C, C-O, C-N, and C-S bonds by means other than hydrolysis or
oxidation.
5. Isomerase: Isomerases just rearrange the existing atoms of a molecule, that is, create isomers
of the starting material.
6. Ligase: Ligases synthesize C-C, C-S, C-O, and C-N bonds in reactions coupled to the
cleavage of high energy phosphate bonds in ATP or some other nucleotide.
Enzyme Kinetics
Michaelis-Menten Equation: vi = (Vmax [S]) / (Km + [S])
vi = the initial velocity - the initial rate of the reaction at a certain substrate concentration
Vmax = the maximal velocity (rate) a reaction can achieve at an infinite concentration of substrate.
Km = Km is the substrate concentration at which the reaction rate is at half-maximum and is a
measure of the substrate's affinity for the enzyme.
A small Km indicates high affinity, meaning that the rate will approach Vmax at lower
concentrations of substrate.
[S] = substrate concentration (the rate of the reaction is dependent on the amount of substrate).
(See figure 9.2 Marks Medical Biochemsitry)
Glucokinase vs hexokinase:
Glucokinase and hexokinase are isozymes. They catalyze the same enzymatic reaction.
Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical
reaction.
Hexokinase is a Michaelis-Menten enzyme; it yields a rectangular hyperbola when the initial
velocity is plotted versus various glucose concentrations.
Glucokinase of liver or pancreas is not a Michaelis-Menten enzyme. It yields a sigmoidal (Sshaped) curve when the initial velocity is plotted versus various glucose concentrations. A
MichaelisMenten graph is not a sigmoidal(S-Shaped) graph and does not apply to allosteric
enzymes

Enzyme regulation
Inhibitors:
Competitive inhibitor: binds in the active site
o Km increases-The substrate concentration has to be higher to compete with the
competitive inhibitor.
o Vmax remains same-If the concentration of substrate is high enough, there is very little
chance for the competitive inhibitor to bind to the active site.
o When a competitive inhibitor is present, it is also binding the active sites so, for any
substrate concentration, the enzyme reaction is slower.
o Can be overcome by an increase in substrate concentration
Noncompetitive inhibitor: binds in a alternative location to the active site
o Vmax decreases
o Km remains same.
o When a noncompetitive inhibitor is present, it binds so strongly that no amount of
substrate can remove it. It is as if the enzyme bound to noncompetitive inhibitor is no
longer there.
See Lineweaver Burke blots: Figure 9.14 Marks Medical Biochemistry
Allosteric effectors: (See figure 9.6 Marks Medical Biochemistry)
o Allosteric activators or positive allosteric effectors or modulators enhance an enzyme reaction
o Stabilize a conformation of the protein that increases binding of substrate and reaction
rate. (R-State)
o Allosteric inhibitors or negative allosteric effectors or modulators inhibit the enzyme reaction
o Allosteric inhibitors bind to the enzyme at an allosteric site and stabilize a conformation
of the protein that decreases binding of substrate and reaction rate. (T state)
o Allosteric compounds bind to a site that is NOT the active site
Allosteric inhibitors would also be considered non-competitive inhibitors.
Covalent Modifications:
o Phosphorylation by a kinase on the R-groups of serine and tyrosine (and sometimes threonine)
o Phosphorylation can:
o change the conformation and the activity of a protein, or
o create a binding site for proteins with a complementary SH (src homology) domain
o Serine kinases phosphorylate serine and tyrosine kinases phosphorylate tyrosine residues.
o Protein phosphatases are enzymes that hydrolyze the phosphoester bonds of phosphoseryl
and phosphotyrosyl residues (R-groups).
o Dephosphorylation changes the conformation of the protein back to the state it was in
before phosphorylation.
o See Muscle glycogen phosphorylase as an example (Figure 9.8 Marks Medical Biochemsitry)
Protein-Protein Interactions
o Association of proteins can activate or deactivate a protein
o Example: G proteins are modulator proteins in cells that possess GTPase activity that slowly
hydrolyze their own bound GTP to GDP and phosphate. As they hydrolyze GTP, their
conformation changes and the complex they have formed with the target protein disassembles.
(See figure 9.9 Marks Medical Biochemistry)
Cleavage
o Removal of a propeptide or cleavage is often required for activation
o Ensure that a protein is active in the correct tissue or cellular compartment

o Examples: chymotrypsin and insulin