CFB 3: Biochemistry Proteins and Enzymes

The pH in the mouth is slightly _____.

Basic

Mixtures of weak acids and their conjugate bases

Buffers

A simple expression that relates pH, pKa, and buffer concentration

Henderson-Hasselbalch equation

Weak acids and bases have characteristic equilibrium constants (K__) also called acid dissociation constants (K_)

Keq

Ka

pH = pKa + log (conjugate base / weak acid)

Henderson-Hasselbalch equation

At the midpoint of the titration, at which exactly 0.5 equivalent of NaOH has been added, [HA] = ?

[HA] = [A-]

pH = pKa

Proteins are composed of a linear sequence of...

Amino acid residues

There are __ amino acid residues found in proteins

20

Proteins are polymers of what?

Amino acids

Glycine

Nonpolar, aliphatic

Alanine

Nonpolar, aliphatic

Proline

Nonpolar, aliphatic

Valine

Nonpolar, aliphatic

Leucine

Nonpolar, aliphatic

Isoleucine

Nonpolar, aliphatic

Methionine

Nonpolar, aliphatic

Phenylalanine

Aromatic

Tyrosine

Aromatic

Tryptophan

Aromatic

What are the aromatic R groups that absorb UV light?

Phenylalanine

Tyrosine

Tryptophan

Lysine

Positively charged

Arginine

Positively charged

Histidine

Positively charged

Serine

Polar, uncharged

Threonine

Polar, uncharged

Cysteine

Polar, uncharged

Asparagine

Polar, uncharged

Aspartate

Negatively charged

Glutamate

Negatively charged

Proteins are composed of amino acids linked together in a chain via _______ _____.

Peptide bonds

Primary structure

Sequence of amino acids

What must a polypeptide do to be functional?

Fold into the correct structure

How can proteins be unraveled?

Heat or mutation

The hydrogen acceptor is usually:

The hydrogen donor is:

Acceptor: oxygen or nitrogen

Donor: another electronegative atom

Secondary structure

Alpha helix

Beta pleated sheet

What AA is the helix breaker?

Proline

Tertiary structure

The fold

What is the oxygen binder of muscle?

Myoglobin

Quaternary structure

>1 polypeptide

Globular protein can non-covalently associate to form multimeric structures:

Dimers, trimers, tetramers, and greater

What is the classic example of quaternary structure?

Hemoglobin

2 alpha chains, 2 beta chains

What has the smallest AA side chain that allows proteins to form sharp bends and for polypeptide chains to come in close contact to one another?

Glycine

Water-soluble proteins are ____ inside.

Oily

Which AA can oxidize to form a covalently linked dimeric amino acid?

Cysteine

Cysteine and disulfide bonds gives extra _________ for "outside" proteins.

Stability

The cytoplasm of cells is ________.

The outside of cells, including ER lumen and blood plasma, is _________.

Cytoplasm: reducing

Outside: oxidizing

Proteins are sensitive to what?

Temperature and pH

Compounds have intrinsic energy called what?

"Free" energy (G)

A drop in free energy (ΔG) indicates a more ______ situation.

More stable

Spontaneous reactions have a _________ ΔG.

Negative

Reactions that flow in the opposite direction have a ________ ΔG.

Positive

ΔG is sensitive to what?

Concentration of reactants and products

T/F: Enzymes alter equilibrium constant.

False

Enzymes typically show maximal activity at a characteristic pH, the pH ______.

pH optima

Amount of product formed per unit time

Velocity

Initial velocity is where on the curve?

Linear part

What do Km values reflect?

Affinity of th eenzyme for the substrate

Double-reciprocal plot

Plot of 1/V vs 1/[S]

Lineweaver-Burke plot

The action of fast-acting enzyme inhibitors depends on what?

How the interact with the enzyme

Competitive inhibitors affect only the what?

Binding site only

Noncompetitive inhibitors affect only the what?

Catalytic machinery

Competitive inhibitors resemble the __________.

Substrate

Example of competitive inhibition

Ethylene glycol for ethanol

What happens to Vmax and Km in the presence of a competitive inhibitor?

Vmax: same

Km: increases

Do non-competitive inhibitors compete with substrate binding?

No

T/F: Inhibitors showing non-competitive kinetics can be reversible or irreversible.

True

What happens to Vmax and Km in the presence of a non-competitive inhibitor?

Vmax: decreases

Km: same

Mechanism of serine proteases

Chymotrypsin, a digestive enzyme, catalyses peptide bond hydrolysis

Enzymes contain an active site, which is usually a crevice on the surface of the enzyme

Do allosteric enzymes contain multiple subunits?

Usually yes

Do allosteric enzymes usually follow Michaelis-Menten kinetics?

No

Allosteric enzymes usually have binding sites for what?

Effector molecules (bind somewhere else that speed things up, or slows them down)

Why are enzymes remarkable catalysts?

Activation energy lowering by transition state stabilization

What is Km and Vmax?

Km: measures substrate affinity

Vmax: speed of enzyme preparation

What is meant by allosteric enzymes?

Activity controlled by effectors

How does a serine protease cleave a substrate?

Specific binding, reactive serine, hydrolysis by water

Consider lidocaine, which is used as a local anesthetic to numb the oral cavity during certain dental procedures. It has a pKa of 7.8. What is the net charge on the molecule at pKa 7.8?

+0.5 (half charged base, half uncharged)

Consider lidocaine, which is used as a local anesthetic to numb the oral cavity during certain dental procedures. It has a pKa of 7.8. What is the ratio of the uncharged form to the charged form at pH 5.8?

0.01

The acid form has a charge of +1, the base form is neutral. You have 100x more acid form than base form, or 0.01 more base form than acid form

What is the least important buffering component of saliva?

Amino acids

Very little "free" AA in the oral cavity; they are polymerized in the form of proteins

Given that the [H+] concentration of an aqueous solution is 10^-4 M, which pH is correct?

4

The pKa of the bicarbonate buffer system (conjugate acid is H2CO3 and base is HCO3-) is 6.1. The pH of a sample of saliva was found to be 7.1. What is the ratio of HCO3- to H2CO3?

10:1

More base than acid because above the pKa

Phosphoric acid has three pKas (pK1: 2.1, pK2: 7.1, pK3: 12.3). The buffering capacity is least at pH:

4

Which ever is furthest from a pKa

Myoglobin is a monomeric protein present in muscle that binds oxygen. It has all of the following elements of protein structure except which?

Quaternary

Which of the following AAs has a side chain that can participate in hydrogen bonding in proteins?

Asparagine (contains electronegative atoms)

Presence of which AA would break an alpha helix?

Proline

Which polypeptide chain has AA side chains that are capable of hydrophobic interactions?

Leucine-valine-phenylalanine

Which polypeptide chain has AA side chains that are capable of forming hydrogen bonds?

Aspartate-lysine-serine

An individual has a mutation in the binding site of an enzyme such that the substrate binds weakly. What is the most likely change in the enzyme parameters?

Km increases

An individual has a mutation in the non-coding part of DNA such that the enzyme is produced with any change in the primary structure, but only 10% is present. What is the most likely change in the enzyme parameters?

Vmax decreases (because there's less enzyme)

The kinetics of most reactions catalyzed by enzymes can be described in terms of Km and Vmax. Which describes Km?

Concentration of substrate required to give half-maximal velocity

The addition of a noncompetitive inhibitor to an enzyme-catalyzed reaction leads to

Decrease in observed Vmax

The addition of a competitive inhibitor to an enzyme-catalyzed reaction leads to

Increased Km but no change in Vmax

Km is generally considered to be a measure of the

Affinity of enzyme for substrate

The effect of a competitive inhibitor on a Lineweaver-Burke (double-reciprocal) of an enzyme would be to

Decrease the magnitude of the X-intercept

(1/Km gets smaller because Km gets larger)

(The Y intercept is unchanged and the slope gets steeper)