Henderson-Hasselbalch

BMB 460 - Spring 2016- group 1

What is the Hendersen-Hasselbalch equation? pH=pka +

log ([A-]/[HA])
What are its components?
pH= -log of the relative amount of free H and OH ions in a
solution
pka= -log of acid dissociation constant
[A-]= concentration of conjugate base in a solution
[HA]= concentration of weak acid in a solution

Acetic Acid-Acetate Pair as a Buffering System

Buffering action is the

consequence of two reversible

reactions taking place
simultaneously and reaching
their points of equilibrium as
governed by their equilibrium
constants, Kw and Ka.
Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008). Lehninger principles of biochemistry (6th ed.). New York: W.H. Freeman.

any weak acid

HA

corresponding

Various Uses of the Henderson-Hasselbalch Equation

1) Calculating pH with known weak Acid/ conjugate base ratio
2) Calculating
A- / HA ratio with known pH and pKa
a) Ratio of HA to Conjugate Base
b) Ratio of Salt to Acid
c) Ratio of Ionized and Un- ionized forms of the molecule of
interest
3) Determining level of ionization of amino acid side chains based
on pKa and conditions

Relevance & Application

Buffers needed in pH sensitive situations, such as biological
systems.
pH- buffers affect:
Charge of proteins (Amino Acid side chain ionization)
Charge of DNA/RNA (Phosphate backbone)
Enzymes (have an optimal pH)
Applications:
Calculating pH of a solution
Determining buffer concentrations
Calculating ionized/un- ionized concentrations
Calculating pKa using pH

Problem 1
Calculation of pH from Molar Concentrations
What is the pH of a solution containing 0.12M of NH4Cl and 0.03M of NaOH (pKa
of NH4+/NH3 is 9.25)?
pH = pKa + log([A-]/[HA])

Problem 2

Problem 3
Amino acid ionization: Calculate the fraction of histidine that has its imidazole side chain
protonated at pH 7.3. The pKa values for histidine are pK1=1.8, pK2 (imidazole)=6.0, and pK3 = 9.2.
pH = pKa + log([A-]/[HA])
7.3 = 6.0 + log([His]/[HisH+])
1.3 = log([His]/[HisH+])
antilog(1.3) = ([His]/[HisH+]) = 20
His = 20/21 = 0.952 = 95.2%
unprotonated

( 20:1 His/HisH+ )

HisH+ = 1/21 = 0.0476 = 4.8%

protonated

If you are having trouble with these problems and/or still

need more of a review, see section 2.3 of the book (pg. 63).
Recommended practice problems: Chapter 2, #9, #12, #18,
#22, #31, and #32. --- try: 7 and 27 6th edition

Amino Acids
Review

What is an Amino Acid?

alpha Carbon

Amine Group

Carboxylic Acid Group

Variable R Group

Amino Acids (think at neutral pH)

Nonpolar R Groups :

Gly, Ala, Pro, Val, Leu, Ile, Met

Aromatic R Groups:

Phe, Tyr, Trp (F,Y,W) -absorb at 280nm

Polar, Uncharged R Groups:

Ser, Thr, Cys, Asn (N), Gln (Q)

Positively Charged R Groups :

Lys (K), Arg (R)- sometimes His,

Negatively Charged R Groups:

Asp (D), Glu (E)

www.neb.com

Why does R group chemistry matter more for

enzymes?

Can you draw glycyl-glycine?

Most common Amino Acids

involved in enzyme chemistry
and R group:

Hydroxyl (-OH) ST and

tyr (Y)
Thiol (-SH) - C
Carboxylic acid COOglu (E) and asp (D)
Amino -NH3+

lys (K)
Imidazole pKa =6.0
H
ONLY residue buffer at 7

Nucleophilic Functional Groups

(e- donors)
Negatively charged oxygen
Negatively charged sulfhydryl
Carbanion
Uncharged amine group
Imidazole
Hydroxide ion

Functional groups
act as proton
donors or acceptors
in the active sites of
enzymes to help
catalyze chemical
reactions in
biological systems.

Electrophilic Functional Groups (e- acceptors)

Carbon atom of carbonyl group
Protonated imine group
Phosphorous of a phosphate group
Proton

Common Reactions Involving Amino Acids

Oxidation-Reduction Reactions: require COENZYMES amino acids not good
to oxidize/reduce
Many of these reactions involve the loss of two electrons and one or two hydrogen ions..

Group Transfer Reactions:

Transfer of acyl, glycosyl and phosphoryl groups are common. This reaction
occurs through the addition of a nucleophile, ex. hydroxyl group, to an
electrophile such as a carbonyl carbon. An example of an enzyme that
catalyzes this type of reaction is a kinase.

Common Reactions Involving Amino Acids

Aldol and Claisen Condensations and Decarboxylations:
These reactions would be impossible without the environment created from the functional
groups. Electronegative atoms such as oxygen and nitrogen can stabilize

required intermediates.
Aldol condensations occur by the nucleophilic alpha carbanion, stabilized
by an adjacent carbonyl group, attacking a electrophilic carbonyl carbon.
Claisen condensations are similar except the alpha carbanion is stabilized by
an adjacent thioester.
Decarboxylations occur through the formation of an alpha carbanion also
stabilized by an adjacent carbonyl group.

Relation between pka and pH

For each functional group in an amino acid, when:
pH>pKa the functional group becomes deprotonated.
EXAMPLES?

pH<pKa the protons bind to the functional group.

EXAMPLES?

Titration Curves for Amino Acids with non-ionizing R

Amino acids have a zwitterionic form
(dipolar ion) which allow them to act as
either an acid or base at different pH
values
pI (Isoelectric point): pH where the net
charge of the amino acid is 0.
pKa: The pH at which the rate of
protonation of the R group is in equilibrium.

Titration Curves for Amino Acids with ionizing R

Histidine is of interest, because
its ionizable side chain has a
pKa near neutral.
At biological pH, Histidine can be
positively charged or uncharged.

Facilitates enzyme-catalyzed
reactions by acting as a
proton donor or acceptor.

Essentials of Enzymes
By: Kevin, Remy, Fufei, Stephen, Amy, Ben

Enzyme (E): a biological catalyst that increase reaction rates without

being consumed in the reaction (While usually proteins, a small group of
RNA based enzymes called ribozymes also exist)

Substrate (S): a molecule that binds to an enzyme and undergoes a

reaction

Active site: the location (usually a groove/cleft or pocket) on an enzyme

that binds the substrate and contains the key Catalytic residues that
carry out the chemistry - catalyze chemical transformation of S to P
E + S ES EP E + P

Enzyme-catalyzed reactions
Enzymes increase the RATE of the
reaction.

G unchanged Activation energy decreases.

through noncovalent interactions
between E and S

Binding energy: the free energy that is

released by the formation of weak
interactions between a complementary
substrate and enzyme.

Figure 6.3

Enzyme-Substrate interactions

Figure 6.5

Figure 6.4

ENZYMES require native protein structure- Proper primary, secondary,

tertiary, and sometimes quaternary structure formation are critical to enzymatic
activity.

Cofactors: inorganic ions (Fe2+) or CoENZYMES - a complex organic/

metal-organic molecule known as a coenzyme (Biocytin) may be required

Regulatory enzymes: increased/decreased activity in response to certain signals

and display a sigmoid model, reflecting cooperative interactions. (do not follow
Michaelis Menten equation but can be related by Km0.5)

Allosteric enzymes: function by noncovalent binding to regulatory compounds

such as metabolites/cofactors & differ by containing active and regulatory binding
sites
Example : ATCase, Amino acid residue modification - Phosphorylation

Enzyme Kinetics- what assumptions were required to derive MM equation?

V = Vmax[S]/Km + [S]
V: initial velocity
Lineweaver-Burk
Michaelis-Menten
Vmax: maximum velocity
[S]: initial substrate concentration
Km: Michaelis- Menten rate constant, or the rate of breakdown to reactants &
products/formation of enzyme substrate complex
From this equation: Km = [S] when V = 1/2Vmax
At low [S], V is linearly dependent on [S], at high [S] V=Vmax, there is a plateau
and the enzyme is saturated
Kcat = Vmax[Et] - Kcat: turnover number, or the amount of substrate
converted to product when an enzyme is saturated (per unit of time)

Vo = Vmax[S]/Km + [S]
Vmax = kcat [Et]
Km = kcat+k-1
k1

When Kcat is truly rate

limiting (very small
relative to K-1)
Km k-1/k1

Enzyme Efficiency?
Substitute for Vmax = Kcat[Et]

Vo = kcat [Et] [S]/Km + [S]

When [S] is <<< than Km?
Vo = kcat [Et] [S]
Km

+ modulator
- modulator

Bioenergetics
Group-4

Bioenergetics
The study of energy transformations in living systems.
The means by which energy from fuel metabolism (or light
capture) is coupled to a cells energy requiring reactions.

Enthalpy and Entropy

H is enthalpy, the heat contained in a system
-H is exothermic, heat exits the system.
+H is endothermic, heat enters the system
S is entropy, a quantitative expression of randomness in a
system.

Gibbs Free Energy

G = H TS
= G (Final State) G(Ini9al State)
Its a measure of the distance of a system
from its equilibrium

Gibbs Free Energy

G = H TS
= G (Final State) G(Ini9al State)
What G tells: The direc9on of the reac9on(s):
which is more favorable
What G doesnt tell: 1. The pathway of reac9on(s)
2. The speed of reac9on(s)

G (kJ/
mol)

Enzymatic reactions of glycolysis actual free

energy change

Free energy change predicts Direction but

NOTHING about RATE or pathway

Gibbs Free Energy

The energy available to do work in a reaction during a
constant temp and pressure.
Keq

Then the Reaction

>1

<0

Proceeds Forward

Is at Equilibrium

<1

>0

Proceeds in Reverse

Keq is ([C]C[D]D/[A]A[B]B) when the reaction is at equilibrium.

G vs G
Chemical reactions proceed spontaneously until equilibrium is
achieved.

G=G+RTln([C]C[D]D/[A]A[B]B)
G: the biochemical standard free energy change, is a
constant given reaction and set of conditions
G is the free energy available from the reaction at the
substrate and product concentration actually occurring in
the system.

Coupling reactions
G = -7.3 kJ/mol

glucose 1 phosphate

glucose 6 phosphate

G = -1.7 kJ/mol
fructose 6 phosphate

glucose 6 phosphate

G = ? kJ/mol
Glucose 1 phosphate

fructose 6 phosphate

Using [product]/[reactant] concentration

to drive a reaction
G = G+ RT ln [C][D]/[A][B]
G = +1.7 kJ/mol
glucose 6 phosphate

fructose 6 phosphate

To achieve G = - 3.4 kJ/mol

[fructose 6 phosphate]
[glucose 6 phosphate]

= ???

NTP-Nucleoside Triphosphate
Phosphate is a good leaving group and the removal of the
third gamma phosphate has a high -G value.
NTPs are used to transport energy.

Phosphoryl transfer occurs- though

reactions may look like NTP hydrolysis

WHY are NTPs a

preferred option
for energy
storage/use?

Reducing Equivalents
Reducing Equivalent- Single electron equivalent participation
in an oxidation - reduction reaction.
Biological fuel molecules are generally enzymatically
dehydrogenated to lose two equivalent at a time- hydride
ions or two hydrogens
Each oxygen atom can accept two reducing equivalents
passing from substrate to oxygen.