BE 3415 || Exam 3

Enzyme

Catalyst that increases the rate of a reaction

Substrate

S: the reactant

Active Site

the small portion of the enzyme surface where the substrate(s) becomes bound via noncovalent bonds

What are the two models that’ve been developed to describe the enzyme-substrate complex?

Lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape.

Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

Kinetics

The study of reaction rates, or the changes in the concentrations of reactants and products with time.

Reaction Rate

change in concentration of a reactant or product over time.

Rate of a Reaction depends on…

1. Physical states and surface areas of reactants

2. Temperature

3. Solvent and catalyst properties

4. Reactant concentrations

Zero-Order Reactions

• Reaction is independent of concentration

• Common in reactions that are catalyzed by an attachment to a reactive surface

• only a small fraction of the reactant molecules is in a location in which they are able to react. This fraction is continually replenished from the larger pool.

First-Order Reactions

• Reaction is directly proportional to the concentration of one of the reactants

• Can have more than one reactant but other reactants would have to be zero order

Second-Order Reactions

• Reaction rate is proportional to the product of two reactants (two molecules of same or different reactant)

• Either two first order of different reactants or one second order reactant

Temperature dependance of catalysis

Increases rates for chemical reactions until denaturation (protein unfolding) occurs, which will result in a decrease.

Michaelis-Menten Model

• K_m describes

1. the constant derived from other rate constants

2. a measure of how well the enzyme recognize the substrate

3. estimate of the dissociation constant of E from S

• Small K_m means tight binding while high means weak binding

• You can also use this equation to solve for the concentration of the enzyme-substrate complex

V_max

• the theoretical max velocity

• Constant

• to reach this value, it would require ALL of the enzyme molecules are simultaneously bound with the substrate

• approaches asymptotically as substrate is increased

Enzyme Inhibition

• Reversible Inhibitor: binds to an enzyme to inhibit, but can be released

competitive inhibitor: binds to the active/catalytic site and blocks access to it by substrate

noncompetitive inhibitor: binds to anywhere but the active site, inhibiting by changing the conformation of the enzyme

• Irreversible Inhibitor: an inhibitor that can’t be reversed

usually involves formation or breaking of the covalent bonds to or on the enzyme

Feeback Inhibition

• Formation of product that inhibits its own production through the allosteric feedback inhibition

• Helps to sustain a steady or oscillating level of product depending on the kinetics and lifetimes in the pathway

Allosteric Enzymes

• often serve as switches

• biological activity is affected by other substances binding to it, changing the enzyme’s activity the conformation of its structure

• something that modifies the behavior of an allosteric enzyme may be either an allosteric inhibitor or activator.

Non-Allosteric Behavior

• point at which the rate of reaction does not change

• enzyme is saturated

• maximum rate of reaction is reached

Allosteric Behavior

• Sigmoidal shape of rate vs. concentration

• Maximum velocity can be reached, but by a different mechanism

Concerted Model

• Allosteric Enzymes have two conformations

R (relaxed): binds substrate well; the active form

T (tight or taut): substrate easily released; the inactive form

• the presence of substrate shifts the equilibrium from T to R

K_R = [R][S]/[RS] & K_T = [T][S]/[TS]

If R form binds more strongly to substrate, then K_R < K_T

Constant c = K_R/K_T

Sequential Model of Cooperative Binding

• the binding of substrate induces a sequential conformational change from the T to R form

Classification of Enzymatic Reactions

Zymogens

Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into an active enzyme

Ex: Chymotrypsinogen

• stored in pancreas

• when secreted into small intestine, the digestive enzyme trypsin cleaves a 15-unti polypeptide from the original peptide to give pi-chymotrypsin

Enzyme Specificity

• Absolute Specificity: catalyzes reaction of one unique substrate to form a specific product.

• Relative Specificity: catalyzes the reaction of similarly structured substrates to give structurally similar products.

• Stereospecificity: catalyzes a reaction where one stereoisomer is reacted or formed.

requires the binding sites on the enzymes to be asymmetric

Dimers

• In DNA binding proteins, the homodimer properties mean it will recognize the same sequence at two different locations

• Results in the DNA recognition site being an inverted repeat

Restriction Enzymes: Endonucleases

• restrict possibility of attack from foreign DNA

• Type II restriction enzymes: cleave DNA chains at selected sites

• No ATP required

• Recognition sites in dsDNA usually have “palindrome-like” sequence symmetry

Sticky end vs Blunt end by cutting restriction enzymes

• Cleavage can leave staggered “sticky” ends or can produce flat “blunt” ends

Types of restriction sites and frequency

• Blunt Ends: Frequency of occurrence of restriction sites

• Sticky Ends: If assume random DNA sequence has equal amounts of each base (A,T, G, C)

• 5’ Overhang: 6 base cutter / 4 base cutter

• 3’ Overhang: The longer the recognition site is the better the specificity

Cloning

Making many copies of a gene

Why are Plasmids useful to cloning

• Plasmids are naturally occurring non-chromosomal DNA

• Can cleaved by restriction enzymes, leaving sticky or blunt ends

Antibodies

Different Forms of Antibiotics:

Inhibit protein synthesis:

1. Kanamycin

2. Tetracycline

3. Streptomycin

4. Chloramphenicol

5. Erythromycin

Inhibit cell wall synthesis:

1. Ampicillin

Induce DNA breaks:

1. Bleomycin

Cloning Vectors

• Plasmids that can be modified to carry new genes

Examples:

• Replicator (Origin of replication)

• A selectable marker (Antibiotic resistance gene)

• A cloning site

Chimeric Plasmids

• Recombinant plasmids that are hybrid DNA, molecules consisting of plasmid DNA sequences plus inserted DNA elements

• Associated with Non-Directional Cloning

Directional Cloning

• Constructing foreign DNA insert with same set of restriction enzymes

• The resulting insert DNA and vector can only be combined in one direction

Ligation Reaction

Multiple Cloning Region

• Area of the vector that contains many common restriction enzyme sites

• Provides a choice of restriction enzyme sites to use when you cut your DNA of interest and put it in the plasmid

Blue-White Screening

• X-Gal (turns blue when beta glycosidic bond is cleaved)

• Intact lacZ (without an insert) makes a blue color when expressed if X-Gal is present

The plasmid (backbone + biobrick)

• Puts parts together one-by-one

• Unreliable and time consuming but rather easy

• Overhanging the X and S are compatible, when ligated together, they form a new site that can’t be cut from either enzyme (scar)

Polymerase Chain Reaction

• A method to copy a given DNA sequence many times over without bacteria

• Any chosen DNA can be amplified if the sequence on either end is known

• Amplification occurs via oligonucleotide primers

• oligonucleotide primers serve as primers for DNA polymerase and the denatured strands of the large DNA sequence of interest serves as the template.

PCR Animation

• Small sample of DNA can serve as template for DNA polymerase

• Designing complementary primers

• Heating and Cooling Elements: Heating to make template a ssDNA and cooling to let primers bind then increase temperature to start the Taq enzyme

Problems with primers

• Harpin Structure: If the 3’ side binds to 5’ side forms, the 3’ end will be blocked and the primer won’t work

• Primer dimers: Problem if the binding is formed at the 3’ ends

• Primer too long: too high of a melting temperature

• Primer too short: won’t stick to template because not enough to form a complementarity