Henderson-Hasselbalch Equation, Proteins and Enzyme Kinetics

for acidic drugs & molc, charge for acidic functional groups is ________

negative

• charged when unprotonated

charged/protonated form of basic drugs

-NH3+

HH equation

connects the acid dissociation constant with the solution pH.

acid dissociation constant (Ka)

describes the reversible dissociation of an acid (HA) into its conjugate base and the proton

unprotonated acid

charged (negative)

unprotonated base

uncharged (neutral)

protonated acid

uncharged (neutral)

protonated base

charged (positive)

uses of HH eq

o To determine whether the predominant form of a drug is charged or uncharged.

o To determine whether a drug is acidic or basic.

do charged molc. readily diffuse across memb?

no

acidic functional groups generally have a pKa less than…

7

basic functional groups generally have a pkA …

>7

if pH = pKa, what is protonation status?

[protonated] = [unprotonated]

if pH > pKa what is protonation status

[protonated] > [unprotonated]

if pH < pKa, what is protonation status?

[protonated] < [unprotonated]

if pH = pKa, describe charge for acids

[charged] = [uncharged]

if pH > pKa for acids, describe charge status

[charged] > [uncharged]

if pH < pKa, what is charge of acids?

[charged] < [uncharged]

if pH = pKa, what is charge status for bases?

[charged] = [uncharged]

if pH > pKa, what is charge status for bases?

[charged] > [uncharged]

if pH < pKa, what is charge status for bases?

[charged] < [uncharged]

amino acid

small molecules that each contain at least one amino functional group and one carboxylic acid functional group (bound to alpha carbon)

importance of alpha amino & alpha carboxyl groups

allow amino acids to participate in hydrogen bonding & form peptide bonds with one another

peptide bond

formed between the alpha amino group of one amino acid and the alpha carboxylic acid of the next amino acid.

when 2+ amino acids are linked thru peptide bonds, they form ______

small peptides or proteins

proteins are known as ________

polypeptides

what determines the 3D conformation/final structure of a protein?

amino acid sequence

what dictates primary structure?

DNA sequence

N-terminus

amino acid with the only unbound alpha amino group is

located.

C-terminus

the only amino acid in the polypeptide with an unbound alpha carboxyl group

amino acid sequence determines ________ of the polypeptide

properties

what stabilizes secondary structures?

• hydrogen bonds

• hydrophobic interactions

• other interactions

alpha helix

hydrogen bonds form between the oxygen of the alpha carboxyl group of 1 amino acid and a hydrogen from the alpha amino group of another amino acid that is 4 amino acids apart.

beta sheet

amino acid chains are laid down in linear (pleated) strands

can include strands of amino acids from a single polypeptide

chain or from multiple chains that are lined up beside one another.

describe hydrogen bonds in beta-sheet

form between amino acids of different strands (i.e., the amino acids that are involved in each hydrogen bond can be far apart from one another) and at right angles to the sheet.

• As with α-helices, the hydrogen bonds form between the oxygen of the alpha carboxyl group of one amino acid and a hydrogen from the alpha amino group of another amino acid.

highest level of structure that a single polypeptide chain can achieve

tertiary structure

2 important ex. of proteins w quaternary structure

1. hemoglobin

2. immunoglobulin G

the bonds bw proteins & most endogenous/exogenous ligands are ____________

reversible

allosteric modulators

molc. that bind proteins at locations other than the ligand binding site to regulate function of protein

• when bound, they can change the affinity of the protein for the ligand via conformational changes

positive allosteric modulator

will increase the affinity of the protein for the ligand (the protein will want to bind the ligand even more than normal)

• allosteric activators

negative allosteric modulator

will decrease the affinity of the protein for the ligand.

• allosteric inhibitors.

cooperative binding

specific type of allosteric modulation that occurs when multiple binding sites for the same molecule exist on a single protein (such as hemoglobin binding four oxygen molecules).

• When one molecule binds to one of the binding sites, it induces a conformational change in the protein that increases the affinity of the protein for another molecule of the same ligand (i.e., another oxygen molecule) at the other binding sites.

hemoglobin is an example of which type of binding?

cooperative binding

enzyme

protein that accelerates reactions by making the transition state of the reaction more energetically favorable.

esterase function

breaks an ester bond

kinase function

adds a phosphate group to a molecule

transferase function

moves a portion of 1 molc to another molc

decarboxylase function

removes a carboxyl group from a molc

protease function

breaks down a protein

synthase/synthetase function

synthesizes a molc from 2 precursors

phosphatase function

removes a phosphate group from a molc

oxidase function

moves an H from a molc to oxygen to make water or hydrogen peroxide

ligand binding curves describe…

the amount of ligand specifically bound to a protein/enzyme over a range of ligand concentrations, holding the concentration of protein target constant.

what does inflection point on ligand-binding curve indicate?

affinity of the ligand-target interaction

• where 50% of target binding sites are bound to ligand

why is a plateau achieved in a ligand-binding site?

because there are a finite number of binding sites available (a finite number of targets) with which the ligand can bind.

dose-response curve

the amount of specific binding of the drug to the target is equal to the amount of the biological response, which can be graphed on a semi-log plot

• identifies the dose (or concentration) at which the drug produces a half-maximal (50%) response.

EC50

The concentration where 50% of the maximal response is reached is called the effective concentration

agonist

ligand that can specifically bind and activate a receptor (target)

• can be exogenous or endogenous

efficacy of a drug

identified as the maximal response elicited by a drug.

t/f: potency & efficacy are opposites

false

partial agonists

always show lower efficacy than full agonists

• bind to receptor at the active site but only produce a partial biological response even when all available binding sites are occupied

inverse agonists

bind the active site of a receptor that has some basal level of activity, and binding changes the shape of the receptor so the basal activity is lost

antagonists

inhibits the action of a ligand or agonist but has no effect in the absence of the ligand is called an antagonist

• binds either to the ligand/agonist binding site on the receptor or another site such as a regulatory site (allosteric site)

3 steps of enzyme-catalyzed rxn

o Step 1 is when a substrate binds to the enzyme at a specific site on the enzyme (the active site).

o Step 2 is when the substrate is converted to the product, which remains bound to the enzyme.

o Step 3 is when the product is released from the enzyme

cofactors

additional molc that covalently bind and assist the enzyme in carrying out its biological function

V0

initial reaction velocity, or rate at which product forms shortly after the rxn begins

Vmax

maximal rxn rate or rate at which the product forms shortly after the rxn begins

Km can be used as an inexact measure of ________

affinity

high Km indicates _____ affinity

low

lineweaver-burk plot

transformation of the ligand binding curve

• can be used to determine the Km and Vmax

• can identify what type of enzyme inhibitor is interfering w the rxn, when appropriate

2 classes of inhibitors

1. irreversible

2. reversible

irreversible inhibitors

bind covalently to the enzyme or bind tightly in a non-covalent fashion

reversible inhibitors

bind temporarily to the enzyme

3 main types of reversible inhibition

1. competitive

2. uncompetitive

3. mixed & noncompetitive

competitive inhibition

occurs when the inhibitor binds at the same location as the substrate, blocking substrate binding (i.e., competing for the active site)

• forms EI complex

describe Km in competitive inhibition

increases

• because it takes more of the ligand to be able to outcompete the inhibitor for binding to the active site

describe Vmax in competitive inhibition

doesn’t change in the presence of a competitive inhibitor because increasing the substrate concentration eventually eliminates the effect of a competitive inhibitor.

uncompetitive inhibitor

preferentially binds the ES complex to form an ESI complex, rendering the complex catalytically inert

describe Km in uncompetitive inhibition

decreases

describe Vmax in uncompetitive inhibition

decreases

mixed & noncompetitive inhibition

occurs when the enzyme binds at a site close to, but different from, the active site.

• inhibitor will bind regardless of whether the substrate has bound the enzyme

describe Km in mixed & noncompetitive inhibition

increases

describe Vmax in mixed & noncompetitive inhibition

decreases

pure noncompetitive inhibition

inhibitor will bind to a site that is far from the active site, without affecting the Km of the enzyme (since the inhibitor binds far from the active site, it does not affect substrate binding and Km and affinity are unchanged).

o Binding the enzyme will result in a decrease in the Vmax, as shown in the figure below.

o This type of inhibition is rare.