BIOG 1440 Lecture Notes - Lecture 4: Metabolic Pathway, Ionic Strength, Activation Energy

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ivoryscorpion883

6 Feb 2018

School

Cornell University

Department

Biology: General Courses

Course

BIOG 1440

Professor

Shapleigh

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Document Summary

1: enzymes speed up metabolic rxns by lowering energy barriers. If a mechanism exists, the rxn will happen a: free energy increases for reactants to reach transition state (activation energy, does not affect free energy change of reaction. Active site binding critical to lowering activation energy: active site: region on enzyme where substrate binds. Enzymes have optimal conditions: enzyme"s activity can be affected by , general environmental conditions i. 1: optimal temp for typical enzyme=37 c ii. Ex: sulfonamides (antibiotics) i: competitive inhibitors of folate synthesis in bacteria, dpp+paba product i. Increase amounts of paba to outcompete with sulfa: mutate protein that sulfonamide targets so drug binds w/ lower affinity. Express transporter that can pump the drug out. Allosteric regulation of enzymes: occurs when regulatory molecule binds to protein at one site and affects protein"s function at another site, may either inhibit or stimulate an enzyme"s activity, activator stabilizes active form of enzyme.

Related Questions

What are the correct answers to the parts of this quiz marked incorrect? (that is my one question. Please take your time. I value accuracy over speed. )

IncorrectQuestion 2

0 / 1 pts
Studies done by Buchner showed that ground-up yeast cells were able to convert sugar to alcohol. The components of the mixture that were responsible for this transformation were

DNA molecules.

enzymes.

lipids.

carbohydrates.

Bloom's Level: 2. Understand
Learning Outcome: 06.01
Section: 06.01
Topic: History of Microbiology

IncorrectQuestion 3
0 / 1 pts
Bacteria that can absorb light energy and convert it into ATP are commonly called

cytochromes.

lysosomes.

chemotrophs.

phototrophs.

cytochromes AND chemotrophs.

Bloom's Level: 1. Remember
Learning Outcome: 06.02
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 4
0 / 1 pts
The readily usable energy currency of cells is

electricity.

the electron transport system.

ATP.

CTP.

the electron transport system AND CTP.

Bloom's Level: 1. Remember
Learning Outcome: 06.02
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 5
0 / 1 pts
NAD in its reduced form is symbolized as

NADH.

NAD.

NAD-.

NADP.

Bloom's Level: 1. Remember
Learning Outcome: 06.03
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 6
0 / 1 pts
The most common starting pathway for the breakdown of sugars is

respiration.

fermentation.

glycolysis.

oxidation.

Bloom's Level: 2. Understand
Learning Outcome: 06.04
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 7
0 / 1 pts
Which of the following processes generates the greatest amount of energy?

fermentation

aerobic respiration

the Entner-Duodoroff pathway

glycolysis

fermentation AND the Entner-Duodoroff pathway

Bloom's Level: 2. Understand
Learning Outcome: 06.05
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 8
0 / 1 pts
Fermentation

uses an inorganic molecule as the final electron acceptor.

uses an organic molecule as the final electron acceptor.

results in the production of a large amount of ATP.

is necessary in some organisms to produce reduced electron carriers.

uses an organic molecule as the final electron acceptor AND is necessary in some organisms to produce reduced electron carriers.

Bloom's Level: 2. Understand
Learning Outcome: 06.05
Section: 06.01
Topic: Microbial Metabolism

IncorrectQuestion 9
0 / 1 pts
The use of the suffix "ase" on a word denotes a(n)

substrate.

product.

enzyme.

electron acceptor.

substrate AND electron acceptor.

Bloom's Level: 1. Remember
Learning Outcome: 06.06
Section: 06.02
Topic: Microbial Metabolism

IncorrectQuestion 10
0 / 1 pts
Enzymes speed up reactions by

raising activation energy.

producing heat.

reducing entropy.

lowering activation energy.

raising activation energy AND reducing entropy.

Bloom's Level: 2. Understand
Learning Outcome: 06.06
Section: 06.02
Topic: Microbial Metabolism

IncorrectQuestion 11
0 / 1 pts
Most enzymes

are generalists and typically recognize a number of different substrates.

are specialists and typically recognize a single substrate.

are active over a wide pH range.

have several active sites.

are generalists and typically recognize a number of different substrates AND have several active sites.

Bloom's Level: 2. Understand
Learning Outcome: 06.06
Section: 06.02
Topic: Microbial Metabolism

IncorrectQuestion 12
0 / 1 pts
Allosteric enzymes

may bind two substrates.

are used to bind to other enzymes.

have an additional binding site that is involved in regulating enzyme activity.

are twice as fast as single site enzymes.

may bind two substrates AND are twice as fast as single site enzymes.

Bloom's Level: 2. Understand
Learning Outcome: 06.09
Section: 06.02
Topic: Microbial Metabolism

Question 13
1 / 1 pts
Feedback inhibition

is a means of regulating the amount of product produced.

often involves the use of allosteric enzymes.

involves inhibiting the last of a series of reactions.

results in raising the activation energy for the reaction.

is a means of regulating the amount of product produced AND often involves the use of allosteric enzymes.

Bloom's Level: 2. Understand
Learning Outcome: 06.10
Section: 06.02
Topic: Microbial Metabolism

IncorrectQuestion 14
0 / 1 pts
Coenzymes are derivatives of

minerals.

proteins.

lipids.

vitamins.

Bloom's Level: 1. Remember
Learning Outcome: 06.07
Section: 06.02
Topic: Microbial Metabolism

Question 15
1 / 1 pts
Environmental factors that may affect enzyme activity include

temperature

pH

salt

age

temperature, pH AND salt

Bloom's Level: 2. Understand
Learning Outcome: 06.08
Section: 06.02
Topic: Microbial Metabolism

Question 16
1 / 1 pts
Most enzymes function best at

acidic pH and high salt concentrations.

basic pH and low salt concentrations.

neutral pH and high salt concentrations.

slightly above pH 7 and low salt concentrations.

Bloom's Level: 2. Understand
Learning Outcome: 06.08
Section: 06.02
Topic: Microbial Metabolism

Question 17
1 / 1 pts
Mercury in mercurochrome inhibits growth

through competitive inhibition.

through a reversible action.

by oxidizing the sulfhydryl groups in cysteine.

by changing the shape of proteins.

by oxidizing the sulfhydryl groups in cysteine AND by changing the shape of proteins.

Bloom's Level: 3. Apply
Learning Outcome: 06.10
Section: 06.02
Topic: Microbial Metabolism

IncorrectQuestion 18
0 / 1 pts
The term precursor metabolite refers to molecules that

activate cell components.

are used in biosynthesis.

result from cell activities.

are present but inactive.

Bloom's Level: 2. Understand
Learning Outcome: 06.11
Section: 06.03
Topic: Microbial Metabolism

IncorrectQuestion 19
0 / 1 pts
Glycolysis

requires oxygen.

produces 3 ATP molecules.

produces 4 molecules of NAD.

may occur under aerobic or anaerobic conditions.

produces 3 ATP molecules AND produces 4 molecules of NAD.

Bloom's Level: 2. Understand
Learning Outcome: 06.12
Section: 06.03
Topic: Microbial Metabolism

IncorrectQuestion 20
0 / 1 pts
The transition step

links glycolysis to the pentose phosphate pathway.

links the pentose phosphate pathway to the Entner-Duodoroff pathway.

links glycolysis to the TCA cycle.

takes place in the matrix of the nucleus.

Bloom's Level: 1. Remember
Learning Outcome: 06.11
Section: 06.03
Topic: Microbial Metabolism

Question 21
1 / 1 pts
The TCA cycle produces

ATP through substrate-level phosphorylation.

NADH.

FADH2.

precursor metabolites.

All of the choices are correct.

Bloom's Level: 1. Remember
Learning Outcome: 06.12
Section: 06.03
Topic: Microbial Metabolism

Question 22
1 / 1 pts
The electron transport system

requires a membrane.

utilizes the nuclear membrane of eukaryotes.

utilizes the mitochondrial membrane of prokaryotes.

generates a concentration gradient of protons.

requires a membrane AND generates a concentration gradient of protons.

Bloom's Level: 2. Understand
Learning Outcome: 06.13
Section: 06.04
Topic: Microbial Metabolism

IncorrectQuestion 23
0 / 1 pts
Which type of phosphorylation does not require a membrane?

Substrate-level phosphorylation

Oxidative phosphorylation

Photophosphorylation

All types of phosphorylation require a membrane.

Bloom's Level: 2. Understand
Learning Outcome: 06.13
Section: 06.04
Topic: Microbial Metabolism

Question 24
1 / 1 pts
Which pairing is incorrect?

A:T

G:C

A:U

A:G

Bloom's Level: 3. Apply
Learning Outcome: 07.01
Section: 07.01
Topic: Genetics

Question 25
1 / 1 pts
RNA is characterized by which one of the following features?

Deoxyribose.

Thymine.

Ribose.

Double-stranded.

Bloom's Level: 1. Remember
Learning Outcome: 07.01
Section: 07.01
Topic: Genetics

Question 26
1 / 1 pts
DNA is characterized by which of the following feature(s)?

Ribose.

Single-stranded.

Deoxyribose.

Thymine.

Deoxyribose AND thymine.

Bloom's Level: 1. Remember
Learning Outcome: 07.01
Section: 07.01
Topic: Genetics

Question 27
1 / 1 pts
Which of the following is/are true of DNA replication?

It starts at the origin of replication.

Nucleotides are added to the 3' end.

It requires an RNA primer to get started.

It utilizes polymerases.

All of the choices are true.

Bloom's Level: 2. Understand
Learning Outcome: 07.03
Section: 07.02
Topic: Genetics

Question 28
1 / 1 pts
Which is true about the RNA transcript?

It is formed using the DNA minus strand as a template.

It has the same 5'-3' orientation as the DNA positive strand.

It is made in short fragments that are then stitched together.

The template starts at the promoter region.

It is formed using the DNA minus strand as a template, it has the same 5'-3' orientation as the DNA positive strand AND the template starts at the promoter region.

Bloom's Level: 2. Understand
Learning Outcome: 07.04
Section: 07.03
Topic: Genetics

IncorrectQuestion 29
0 / 1 pts
How many nucleotides are in a codon?

1

2

3

4

5

Bloom's Level: 1. Remember
Learning Outcome: 07.05
Section: 07.03
Topic: Genetics

Question 30
1 / 1 pts
Which may be or is an RNA molecule?

AGCCTAC

GGGCCCA

GCCCUUA

AGCCTAC AND GGGCCCA

GGGCCCA AND GCCCUUA

Bloom's Level: 2. Understand
Learning Outcome: 07.01
Section: 07.01
Topic: Genetics

IncorrectQuestion 31
0 / 1 pts
Which molecule carries an anticodon?

DNA

mRNA

rRNA

tRNA

Bloom's Level: 1. Remember
Learning Outcome: 07.05
Section: 07.03
Topic: Genetics

Question 32
1 / 1 pts
AUG

is only used as the start codon.

codes for methionine.

determines the reading frame.

is one of the stop codons.

codes for methionine AND determines the reading frame.

Bloom's Level: 2. Understand
Learning Outcome: 07.05
Section: 07.03
Topic: Genetics

IncorrectQuestion 33
0 / 1 pts
The A-site

is found on the RNA polymerase enzyme.

is found on the 30S ribosome.

is found on the 70S ribosome.

is the amino acid site.

is found on the 70S ribosome AND is the amino acid site.

Bloom's Level: 1. Remember
Learning Outcome: 07.05
Section: 07.03
Topic: Genetics

IncorrectQuestion 34
0 / 1 pts
A stop codon

codes for the stop amino acid s-methyl-methionine.

forms a hairpin loop forcing the ribosome to fall off.

codes for no amino acid.

enhances the binding of the e-polymerase.

codes for the stop amino acid s-methyl-methionine AND forms a hairpin loop forcing the ribosome to fall off.

Bloom's Level: 2. Understand
Learning Outcome: 07.05
Section: 07.03
Topic: Genetics

IncorrectQuestion 35
0 / 1 pts
Some segments of the precursor mRNA in eukaryotes are non-coding and are called

exons.

introns.

integrans.

uselessans.

Bloom's Level: 1. Remember
Learning Outcome: 07.06
Section: 07.04
Topic: Genetics

Question 36
1 / 1 pts
Gene regulation may entail

turning on genes only when needed.

turning off genes when not needed.

turning on or off entire groups of genes.

All of the choices are correct.

Bloom's Level: 2. Understand
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

IncorrectQuestion 37
0 / 1 pts
The set of genes in bacteria that are linked together and transcribed as a single unit is referred to as a(n)

operon.

regulon.

operator.

repressor.

Bloom's Level: 1. Remember
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

Question 38
1 / 1 pts
The molecules that bind to a repressor and cause it to no longer bind to the operator are called

activators.

repressors.

introns.

inducers.

Bloom's Level: 2. Understand
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

Question 39
1 / 1 pts
Repressors

are involved in negative control.

are involved in positive control.

always bind to the promoter.

bind or do not bind to the operator depending on their shape (conformation).

are involved in negative control AND bind or do not bind to the operator depending on their shape (conformation).

Bloom's Level: 2. Understand
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

Question 40
1 / 1 pts
Activators

are involved in negative control.

are involved in positive control.

always bind to the promoter.

are allosteric proteins.

are involved in positive control AND are allosteric proteins.

Bloom's Level: 2. Understand
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

Question 41
1 / 1 pts
Signal transduction

is the relay of information about conditions outside a cell to inside the cell.

often relies on a two component system.

may involve phosphorylation of various molecules.

is used by certain pathogens to sense low magnesium conditions.

All of the choices are correct.

Bloom's Level: 2. Understand
Learning Outcome: 07.07
Section: 07.05
Topic: Genetics

IncorrectQuestion 42
0 / 1 pts

Quorum sensing

is used by bacteria to sense the density of cells.

is used by bacteria to sense the proximity of eukaryotic cells

is used by bacteria to limit the density of cells.

is used by eukaryotes to sense the presence of bacteria.

does not influence the expression of genes by a bacteria.

Quorum sensingBloom's Level: 2. UnderstandLearning Outcome: 07.07Section: 07.05Topic: Genetics

Question 43
1 / 1 pts
In DNA sequence analysis

the + strand of DNA is used.

the start codon is ATG.

ORFs are searched for.

codon usage is a helpful indicator for protein coding areas.

All of the choices are correct.

Bloom's Level: 2. Understand
Learning Outcome: 07.12
Section: 07.08
Topic: Genetics

IncorrectQuestion 44
0 / 1 pts
DNA replication is usually unidirectional.

True

False

Bloom's Level: 2. Understand
Learning Outcome: 07.03
Section: 07.02
Topic: Genetics

IncorrectQuestion 45
0 / 1 pts
There are 4 functional types of RNA.

True

False

Bloom's Level: 1. Remember
Learning Outcome: 07.04
Section: 07.03
Topic: Genetics

Question 46
1 / 1 pts
Antisense RNA is the complement of the plus strand and may be useful in inhibiting translation.

True

False

Bloom's Level: 2. Understand
Learning Outcome: 07.11
Section: 07.07
Topic: Genetics

IncorrectQuestion 47
0 / 1 pts
A drug that inhibits the activity of bacterial DNA gyrase ______ be a good antibiotic because _______.

would; it would inhibit all DNA synthesis

would NOT; it would inhibit all DNA synthesis, even our own cell's DNA synthesis

would; it would selectively inhibit bacterial DNA synthesis due to differences between bacterial/human gyrase

would NOT; the DNA replication machinery could still function, even with impaired DNA gyrase enzyme

Bloom's Level: 5. Evaluate
Learning Outcome: 07.03
Section: 07.02
Topic: Genetics

IncorrectQuestion 48
0 / 1 pts
The regulatory protein

binds to the promoter region of DNA.

may inhibit or enhance transcription.

may control translation of the operon.

affects the activity of the DNA polymerase.

binds to the promoter region of DNA AND affects the activity of the DNA polymerase.

Bloom's Level: 2. Understand
Learning Outcome: 07.09
Section: 07.06
Topic: Genetics

Question 49
1 / 1 pts
The source of variation among microorganisms that were once identical is

antibiotic resistance.

virulence factors.

sigma factors.

mutation.

Bloom's Level: 1. Remember
Learning Outcome: 08.02
Section: 08.01
Topic: Genetics

Question 50
1 / 1 pts
The characteristics displayed by an organism in any given environment is its

genotype.

archaetype.

mutatotype.

phenotype.

Bloom's Level: 2. Understand
Learning Outcome: 08.01
Section: 08.01
Topic: Genetics

Question 51
1 / 1 pts
Transposons

are informally known as jumping genes.

may cause insertion mutations.

may cause knockout mutations.

were first recognized in plants.

All of the choices are correct.

Bloom's Level: 2. Understand
Learning Outcome: 08.05
Section: 08.02
Topic: Genetics

Question 52
1 / 1 pts
The largest group of chemical mutagens consists of

radiation.

base analogs.

nitrous acid.

alkylating agents.

Bloom's Level: 1. Remember
Learning Outcome: 08.06
Section: 08.03
Topic: Genetics

Question 53
1 / 1 pts
Nitrous acid most frequently causes mutations by

substituting oxygen for hydrogen bonds.

converting keto groups to amino groups.

altering the bonding ratios of nitrogen bases.

converting cytosine to uracil.

Bloom's Level: 2. Understand
Learning Outcome: 08.06
Section: 08.03
Topic: Genetics

IncorrectQuestion 54
0 / 1 pts
Chemical mutagens that mimic the naturally occurring bases are called

nitrogen mustards.

alkylating agents.

base analogs.

nitrous oxide.

Bloom's Level: 1. Remember
Learning Outcome: 08.06
Section: 08.03
Topic: Genetics

IncorrectQuestion 55
0 / 1 pts
Irradiation of cells with ultraviolet light may cause

4 nucleotides to covalently bind together.

thymine dimers.

adenine complementary base pairing with cytosine.

the addition of uracil.

Bloom's Level: 2. Understand
Learning Outcome: 08.08
Section: 08.03
Topic: Genetics

Question 56
1 / 1 pts
Thymine dimers are dealt with by

no repair mechanisms.

photoreactivation repair.

SOS repair.

excision repair.

photoreactivation repair AND excision repair.

Bloom's Level: 2. Understand
Learning Outcome: 08.11
Section: 08.04
Topic: Genetics

IncorrectQuestion 57
0 / 1 pts
X-rays

have no effect on DNA.

cause thymine trimers.

cause single and double strand breaks in DNA molecules.

make the DNA radioactive.

Bloom's Level: 1. Remember
Learning Outcome: 08.08
Section: 08.03
Topic: Genetics

IncorrectQuestion 58
0 / 1 pts
DNA repair mechanisms occur

only in prokaryotes.

only in eukaryotes.

in both eukaryotes and prokaryotes.

in neither eukaryotes or prokaryotes.

Bloom's Level: 1. Remember
Learning Outcome: 08.09
Section: 08.04
Topic: Genetics

IncorrectQuestion 59
0 / 1 pts
Antibiotics

cause mutations to occur.

may act as alkylating mutagens.

provide an environment in which pre-existing mutants survive.

increase the rate of spontaneous mutation.

Bloom's Level: 2. Understand
Learning Outcome: 08.12
Section: 08.05
Topic: Genetics

IncorrectQuestion 60
0 / 1 pts
Prokaryotic cell mutations can be observed very quickly because the prokaryotic chromosome is

diploid.

polyploid.

haploid.

polysomal.

Bloom's Level: 2. Understand
Learning Outcome: 08.02
Section: 08.01
Topic: Genetics

Question 61
1 / 1 pts
The diploid character of eukaryotic cells may mask the appearance of a mutation since

this may be a frame shift.

the mutation is often reversible.

the mutation may be palindromic.

the matching chromosome may carry the dominant gene.

Bloom's Level: 2. Understand
Learning Outcome: 08.02
Section: 08.01
Topic: Genetics

IncorrectQuestion 62
0 / 1 pts
A clever technique that streamlines the identification of auxotrophic mutants is

gas chromatography.

replica plating.

direct selection.

reversion.

Bloom's Level: 2. Understand
Learning Outcome: 08.12
Section: 08.05
Topic: Genetics

IncorrectQuestion 63
0 / 1 pts
To increase the proportion of mutants in a population of bacteria one may use

direct selection.

replica plating.

penicillin enrichment.

individual transfer.

Bloom's Level: 2. Understand
Learning Outcome: 08.12
Section: 08.05
Topic: Genetics

Question 64
1 / 1 pts
Bacteria that have properties of both the donor and recipient cells are the result of

UV light.

SOS repair.

frame shift mutations.

genetic recombination.

Bloom's Level: 2. Understand
Learning Outcome: 08.13
Section: 08.06
Topic: Genetics

IncorrectQuestion 65
0 / 1 pts
The mechanism by which genes are transferred into bacteria via viruses is called

ellipsis.

replica plating.

transformation.

transduction.

conjugation.

Bloom's Level: 1. Remember
Learning Outcome: 08.14
Section: 08.07
Topic: Genetics

IncorrectQuestion 66
0 / 1 pts
In conjugation the donor cell is recognized by the presence of

an F plasmid.

a Y chromosome.

diploid chromosomes.

an SOS response.

an F plasmid AND diploid chromosomes.

Bloom's Level: 2. Understand
Learning Outcome: 08.15
Section: 08.08
Topic: Genetics

IncorrectQuestion 67
0 / 1 pts
Gene transfer that requires cell-to-cell contact is

transformation.

competency.

conjugation.

functional genomics.

Bloom's Level: 2. Understand
Learning Outcome: 08.15
Section: 08.08
Topic: Genetics

Question 68
1 / 1 pts
The transfer of vancomycin resistance from Enterococcus faecalis to Staphylococcus aureus is thought to have involved

conjugation.

transformation.

transduction.

transposons.

conjugation AND transposons.

Bloom's Level: 2. Understand
Learning Outcome: 08.17
Section: 08.09
Topic: Genetics

IncorrectQuestion 69
0 / 1 pts
Double-stranded DNA enters the recipient cell during transformation.

True

False

Bloom's Level: 2. Understand
Learning Outcome: 08.13
Section: 08.06
Topic: Genetics

Question 70
1 / 1 pts
Plasmids often carry the information for antibiotic resistance.

True

False

Bloom's Level: 2. Understand
Learning Outcome: 08.16
Section: 08.09
Topic: Genetics

Quiz Score: 31 out of 70

describe the definitions of substrate, enzyme active site and its general characteristics, and apoand

holo-enzymes.

Â· describe the fact that enzyme catalysis is specific in terms of the type of reaction and the exact

substrate structure.

Â· Explain the difference in the âlock-and-keyâ model and the induced fit model of enzyme

substrate interaction.

Â· describe that reaction rate constant thus reaction rate is dependent on the activation energy.

Â· describe the relationship between the energy of the transition state and the activation energy.

Â· describe the relative binding affinity of a substrate and its transition state to the enzyme.

Â· Explain how enzymes speed up reaction rate, i.e., how do enzymes lower the activation

energy. Note: Factors other than preferential stabilization of transition state also contribute to increased

reaction rate. They include 1) enzymes placing one substrate (reactant) next to the other substrate (reactant)

so that the reaction is no longer dependent on the collision rate of the two substrates; 2) enzymes sequester the

substrate(s), minimizing solvent competition thus speeding up the reaction; 3) in enzyme complexes with each

enzyme catalyzing one step in a multi-step reaction sequence, the product of one reaction is fed to the active site

of another enzyme for the next step without the product diffusing away from the enzyme complex thereby

increasing reaction rate.

Â· describe the Michaelis-Menten equation relating the initial velocity to total substrate

concentration.

Â· describe the assumptions that went into the derivation of the Michaelis-Menten equation. ([E]

is constant, steady state assumption, etc)

Â· describe the relationship between Vmax and kcat and the fact that kcat is a constant for a given

enzyme and substrate at a given temperature and solution condition; whereas Vmax can differ

for a given enzyme and substrate at a given temperature and solution condition, depending

on the enzyme concentration.

Â· describe the definition of Kd (equilibrium dissociation constant for ES into E and S).

Â· describe the relationship between Kd and the affinity of substrate for the enzyme, i.e., the

higher the Kd, the weaker the binding between E and S.

Â· describe the relationship between Kd and Km, i.e., in general, the higher the Kd, the higher theKm.

Â· Use the Michaelis-Menten equation and the relationship between Vmax and kcat to do

calculations.

Â· describe the other names for the catalytic rate constant, kcat (i.e., the turn over number; also

called krelease) and why it is so called (kcat reports on per unit time per enzyme molecule the

number of product molecules formed/released, thus the name turnover number or krelease).

Â· Determine Vmax and Km using both the Michaelis-Menten plot (initial velocity versus [S]) and

the Lineweaver-Burk Plot (the double reciprocal plot), given the data of initial velocity and

[S].

Â· Draw the Michaelis-Menten plot AND the double reciprocal plot (Lineweaver Burk), given

the Vmax and Km values.

Â· describe the fact that the term kcat/Km is the best parameter for measuring the catalytic

capability (efficiency) of an enzyme.

Â· describe the existence of irreversible enzyme inhibition and the general mechanisms of

irreversible inhibition.

Â· describe the different types of reversible inhibitions, namely the competitive, uncompetitive,

and mixed inhibitions (noncompetitive inhibition is a special case in which the inhibitor

binds E and ES with equal affinity therefore Î± is equal to Î±â â Km is not affected).

Â· Describe each type of inhibition in terms of inhibitor binding site (active site or other than

the active site), how the inhibitor affects vmax and Km and the quantitative relationship of

each parameter with and without the inhibitor present and their relationship with [I] and KI

or KI

Â· Show the characteristics of each type of inhibition on a Michaelis-Menten plot (initial

velocity versus total substrate concentration), i.e. draw the curves with and without the

competitive inhibitor.

Â· Show the characteristics of each type of inhibition on a double reciprocal plot (LineweaverBurk

Plot) by drawing the lines with and without the competitive inhibitor.

Â· Given the Michaelis-Menten or double reciprocal plot with and without the inhibitor,

determine the type of inhibitor.

Â· describe the fact that transition state analogs are potent inhibitors.

Â· Explain why transition state analogs are potent inhibitors.

Â· describe that not all enzymes obey Michaelis-Menten kinetics.

Â· describe situations when an enzyme will not obey Michaelis-Menten kinetics (when the enzyme

catalyzes reactions with multiple substrates or when the enzyme is allosteric, i.e. binding of

the substrate at one active site affects the other active site(s) for the same substrate).

Â· Name the four types of catalytic mechanisms. (electrostatic catalysis, general/specific acid

base catalysis, metal ion catalysis, covalent catalysis)

Â· Contrast acid/base catalysis to general acid/base catalysis, i.e., comment on their similarity

and difference.

Â· Name the eight common general acid/base pairs in proteins and write the formula of the

acid and base form for each pair (hint: consider the eight amino acids that have ionizable

side chainsâ¦)

Â· describe the meaning of covalent catalysis.

Â· describe the reaction catalyzed by chymotrypsin and the three catalytic residues in

chymotrypsin.

Â· Draw the interactions among the three catalytic residues (catalytic triad).

Â· State the catalytic role of each of the catalytic residues and how they accomplish that.

Â· describe the steps in the formation of the acyl-enzyme intermediate, and the substrate and

products in this first part of the chymotrypsin mechanism.

Â· Draw the transition state structure for the formation of the acyl-enzyme intermediate and

indicate by drawing how it is preferentially stabilized.

Â· describe the steps in the regeneration of chymotrypsin (deacylation), the substrates and

product of this second part of the chymotrypsin mechanism.

Â· Draw the transition state for deacylation and explain how it is preferentially bound to or

stabilized by the enzyme.

Â· Explain what general acid and base catalysis refers to and what groups act as the general acid

and base in the chymotrypsin mechanism.

Â· Explain what covalent catalysis refers to in the chymotrypsin mechanism.

Â· Explain the basis for the substrate specificity of chymotrypsin (consider the composition of

the active site â chymotrypsin specifically cleaves peptide bonds adjacent to aromatic

residues)

Â· Explain the term âserine proteaseâ. Answer: these are proteases (enzymes that

hydrolyze peptide bonds) that have serine as a catalytic residue (that acts as a

nucleophile, which can attack the carbonyl carbon of the peptide bond).

Â· Key features of Hexokinase, enolase and lysozyme mechanisms that were discussed in class

(specific methods of catalysis utilized by these enzymes; general description of overall

catalytic transformation)

Â· describe the four regulatory strategies of enzyme catalysis

Â· describe the definition of feedback inhibition (mechanism of cellular regulation that regards an

enzyme that catalyzes the production of a given substance that then inhibits the cell when

concentration of that substance has reached an appropriate level â this balances/regulates

amount of product provided with amount needed)

Â· describe the most common type of covalent modification and the types of enzymes

responsible for the addition and removal of the chemical group in this covalent

modification.

Â· Explain the general mechanism of phosphorylation in regulating enzyme activity.

Â· describe the general naming scheme for active and inactive proteases.

Â· describe that protease inhibitors exist to inhibit protease.

Â· describe that enzymes are regulated through covalent and non-covalent processes.

Â· Noncovalent regulation

o Describe noncovalent modification in general terms. Elaborate on the relation

among enzyme activity, allostery, effector molecules (small molecules) and control

proteins.

o Concerning allosteric regulation, some enzymes have multiple active sites for the

same substrate and the binding of the substrate increases the activity of the enzyme

by stabilizing the R (more active) state over the T (relatively inactive) state. (Lecture

13: âNoncovalent Modification: Allosteric Regulatorsâ)

Ã Draw a curve for the initial reaction velocity versus substrate concentration.

Ã describe and explain the specific shape of the v0 vs. [S] plot.

Ã Draw on the same graph above a v0 vs. [S] curve for the enzyme in the

absence of allostery. and how th i.e., in the absence of conformational change

upon substrate binding. Comment on the shape of this curve.

Ã Comment on the benefit of allosteric activation.

Ã When an inhibitor is present for such an allosteric enzyme, the v0 versus [S]

plot will shift. Draw a v0 vs. [S] curve with and without the inhibitor.

Ã When an activator is present for such an allosteric enzyme, the v0 versus [S]

plot will shift. Draw a v0 vs. [S] curve with and without the inhibitor.

Ã When an activator is present for such an allosteric enzyme, the v0 versus.

Ã describe the relationship between feedback inhibition and allosteric regulation

and the reason behind the term âfeedback inhibitionâ.

Â· Covalent regulation

o describe the most common covalent modification, the enzymes that catalyze the

modification and the removal of that modification, respectively and the amino acid

residues that tend to be the site of this modification.

o Write the structure of the added group bonded to those residues and the reaction for

this covalent modification.

o Rationalize the effectiveness of phosphorylation on altering the activity of an

enzyme.

o describe the mechanism of chymotrypsinogen activation to chymotrypsin (cleavage of

zymogen by trypsin).

Summarize the following reading (Nutrient (zinc and vitamin E)-gene interactions related to inflammatory and antioxidant response in aging and inflammation) and it should be at least one paragraph (100-200 words) and include the following information:

* Main point of the reading

* Describe how the health deviations you read about alter normal physiology.

* Provide your key take away from the reading. This could be something that made you pause, changed your thinking, supported your thinking, or made you question something.

INTRODUCTION Aging is an inevitable biological process that is accompanied by gradual and spontaneous biochemical and physiological changes including increased susceptibility to diseases, adverse environmental conditions, and loss of mobility and agility. Alterations in neuroendocrine-immune interactions as well as in antioxidant capacity also play a fundamental role in aging. The inability of an organism to remodel these changes may lead to the appearance of some degenerative age-related diseases. As a result, the remodeling theory of aging has been proposed (1). Various nutritional factors are directly linked to these phenomena, as, for instance, in restoring the neuroendocrine-immune network, metabolic harmony, and the capacity to respond to oxidative stress (2). Approximately 40 micro nutrients (vitamins, essential minerals, and other compounds required in small amounts for normal metabolism) have been reported as essential components in the diet (3). The dietary intake of essential macro nutrients and micronutrients is usually inadequate in the elderly (4). Several causes contribute to this gap. First of all, the poor socio-economic conditions in which a large part of the older population live may lead to a consumption of inexpensive foods deficient in micronutrients, such as carbohydrates (5). The gap is worsened by the loss of appetite, lack of teeth, intestinal malabsorption, and decreased requirements for energy that lead finally to frailty, disability, and mortality (6). Some authors have reported that the deficiency of macro nutrients and micronutrients in aging is strictly related to global impairments of immune functions, metabolic harmony, and antioxidant defense by external noxae with subsequent appearance of age-related diseases (7). Indeed, many micronutrients contribute directly or indirectly to the biological activity of some antioxidant enzymes (e.g., SOD, GPx, and catalase) (8), to the efficiency of the immune system (9), and to the maintenance of metabolic function especially in preventing mitochondrial decay (4). In this last context, feeding studies in older rats have shown that mitochondrial metabolites and antioxidants protect neuronal cells from neurotoxin- and oxidant-induced toxicity and oxidative damage, delay the normal senescence of human diploid fibroblasts, and inhibit the oxidant-induced acceleration of senescence (10). With advancing age, increased oxidative damage to proteins and lipid membranes, particularly in mitochondria, causes a deformation of the structure of enzymes, with a consequent decrease in enzyme activity as well as substrate binding affinity for their substrates. An increased level of the substrate by micro nutrients restores the speed of the reaction as well as mitochondrial function, thus delaying mitochondrial decay and aging (10, 11). In contrast, recent longitudinal studies of dietary daily intake in human nonagenarians and centenarians (successful aging) have shown that an adequate consumption of micronutrients and macronutrients, as well as a satisfactory content of some trace elements within the cells, leads to good performance in several immune functions, metabolic compensation, and preservation of antioxidant activity (12, 13). In this context, polyunsaturated fatty acids, highly sensitive to reactive oxygen species, decrease in liver mitochondria from human centenarians, a feature acquired during evolution as a protective mechanism to favor longevity (14). Therefore, nutritional factors may play a pivotal role for healthy aging and longevity. However, the effects of the nutritional factors are strongly influenced by genetic factors, in particular by the genes involved in inflammatory/immune responses. Proinflammatory cytokines such as IL-1, IL-6, and TNF-Î±; the antiinflammatory cytokine IL-10; the HSP70 chaperones; and the regulators of trace element homeostasis, metallothioneins (MTs), seem particularly relevant, taking into account that the same genes are involved in the susceptibility to major geriatric disease/disorders such as diabetes, osteoporosis, osteoarthritis, dementia, cardiovascular diseases, and infections (15, 16). Indeed this reemphasizes that up to 25% of the variation in human life span is heritable (17); the rest is due to environmental and lifestyle factors, which impact the aging process, contributing as such to a large interindividual variability. Therefore, one current challenge is to understand how the interaction between genetic factors and nutrients may influence aging and longevity, in view of the high impact on gene expression, protein production, and epigenetic mechanisms implicated in regulating the life span (18). Some dietary patterns with likely different impacts on longterm disease occurrence and survival have been identified. In this context, strong evidence for a beneficial effect of higher conformity with the Mediterranean dietary pattern on causes of death, including those of cardiovascular diseases and cancer, has been reported (19). Anyway, it is also commonly accepted that the complex interactions of multiple polymorphisms play a key role in how individuals may respond to dietary interventions (20). For each nutrient, there is a window of intake between the recommended dietary allowance (RDA), which is defined as the dietary intake sufficient to meet the requirements of 97% of healthy individuals in a particular stage of life and sex group, and the tolerable upper limit (UL), which is the highest nutrient intake that can be achieved without incurring a risk of adverse health effects for most individuals in the general population (21). Although worldwide research on genetic variations that require a different RDA or UL is still in progress, several genes and alleles have been suggested to affect nutrient utilization, including genes involved in the metabolism of folate and vitamin B12 (22), vitamin E (23), lipids (24), iron (25), and zinc (26). In this last case, it is important to note that the RDA (11 mg/day for men, and 8 mg/day for women) and UL (40 mg/day for adults) for zinc are very close (Food and Nutrition Board and Institute of Medicine, 2001). Without excluding the beneficial effects of all of these nutrients for healthy aging and longevity (27), but taking into account the pivotal role played by zinc and vitamin E in fighting oxidative stress in aging and in some age-related diseases, such as atherosclerosis (23), we herein report the studies carried out on the interrelationships between zinc and some genes related to the inflammatory/immune response (such as MT and IL-6), as well as the interactions between vitamin E and the gene expressions related to inflammation. Clinical trials of zinc and vitamin E are also reported for their possible beneficial effects on longevity or in preventing the initiation and progression of cardiovascular diseases.

ZINC-MT GENE INTERACTION MTs MTs are essential to intracellular zinc homeostasis by sequestration and release of the metal when it occurs and thereby controlling available free zinc ions (28). The cysteine sulfur ligands in the cluster structure of MT can be reduced (zinc sequestration) or oxidized (zinc release), thus with concomitant changes in the relative amount of bound and free zinc (29). MTs are genetically polymorphous protein families with subfamilies, subgroups, and various isoforms. Humans possess genes for 4 subfamilies (encoded by at least 10 functional MT genes), all located on chromosome 16: the brainspecific MT3, the squamous epitheliumâspecific MT4, and the ubiquitous MT1 and MT2 (30). One of the first functions of MT1 and MT2 is to regulate zinc homeostasis and to limit oxidative damage within the cells (28). Following an injurious stimulus, such as a transient inflammation, the subsequent oxidative stress induces the release of zinc from MT via nitric oxide (NO), to promote the activity and expression of antioxidant enzymes, including MT itself, thus reducing the oxidative damage and the consequences of the injurious stimulus (31). However, the increased expression of proinflammatory cytokines occurring in aging, leads to increased expression of MTs, which in turn sequester considerable amounts of zinc, making it less available for an efficient immune response (32). If, on the one hand, the reduced zinc ion availability in old age might indicate an excessive sequestration of zinc ions by MTs, on the other, consideration of recent findings on oxidative modification of MTs leading to their loss of function suggests also a mechanism whereby these proteins can also lose their ability to buffer the intracellular free zinc concentration (33). In this case, MTs would be unable either to bind or to consequently release zinc in response to stressors. However, it is still unclear if dysfunctional MTs can be considered a typical alteration associated with specific disease/disorders, such as hyperhomocysteinemia and type 2 diabetes, or if they are a common feature of aging. Anyway, as a consequence of the altered zinc metabolism, a large number of genes that act as zinc sensors and transcriptional activators and/or repressors are also altered in aging (34). This may have a deep impact on the regulation of zincdependent transcription factors, because they not only regulate âzinc sensitiveâ genes but also control their own transcription through positive autoregulatory mechanisms (35). Taking into account that healthy centenarians display a low MT expression and satisfactory zinc ion availability despite increases in proinflammatory cytokines (IL-6, TNF-Î±) (36), it may be suggested that this feature reflects the existence of compensatory phenomena able to counteract the effects of inflammation in these exceptional individuals. Moreover, these data suggest that a preservation of zinc homeostasis is an important feature of healthy centenarians. Therefore, the role played by the zinc-gene interaction is pivotal to successful aging and, at the same time, to escape from some age-related diseases. Since, the persistence of inflammatory stimuli over time represents the biological background favoring susceptibility to age-related diseases/ disabilities, the absence of specific ârobustâ gene variants and/or the presence of specific âfrailâ gene variants might predict, on the one hand, longevity, and on the other, the predisposition to the appearance of the more common age-related diseases, such as infections and cardiovascular diseases. In this context, it has to be considered that the genes selected, because they confer a reproductive advantage early in life, may have dangerous effects in the postreproductive period. In fact, negative selection against these harmful effects fails due to the decline of natural selection with age. This fact means that one gene that is âfavorableâ in young/adult age may be âdisadvantageousâ in aging (the antagonistic pleiotropy theory of ageing (37)). Following this perspective, the beneficial effect of inflammation, via an optimal MFT-1-MT-zinc-gene interaction, in young and adult age may become detrimental in old age (32). The recent discovery of novel polymorphisms of MT2A and MT1A supports this assumption. Indeed, older subjects carrying the AA genotype for the MT2A polymorphism display low zinc ion bioavailability, chronic inflammation by high IL-6 and altered lipid assessments, with subsequent elevated risk for atherosclerosis and type 2 diabetes (38). By contrast, a polymorphism corresponding to an A/T (asparagine/ threonine) transition at the +647 nt position in the MT1A-coding region is the most often involved in female longevity (39). Such allelic variants may be very useful tools to screen older subjects at risk for zinc deficiency on a genetic basis, taking into account that the actual methodological procedures to test âzinc statusâ are often misleading and that laboratory investigations to assay zinc ion bioavailability are scarcely reproducible and poorly applicable to clinical practice (40). In this context, a novel reproducible system for testing intracellular zinc ion bioavailability has been developed using a zinc fluorescent probe (Zynpir-1) associated with MT values, with both tests representing valid methods to detect the intracellular zinc status (41).

ZINCâINTERLEUKIN-6 GENE INTERACTION Interleukin-6 (IL-6) is a pleiotropic cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types, and it plays a pivotal role in immune response. In particular, the most important function of IL-6 is most likely as a mediator of acute phase inflammatory responses. These include the balance of the proinflammatory/antiinflammatory pathways, lymphocyte activation, and hepatocellular stimulation of acute phase protein synthesis (42). Studies of the effects of aging on inflammatory response show IL-6 to be an important âcytokine for gerontologistsâ. Recent evidence has shown that abnormally increased concentrations of IL-6 are reliable markers for functional disability and predictors of disability and mortality among the elderly (43). Moreover, IL-6 dysregulation is involved in age-related inflammatory diseases, such as cancer, lymphoma, cardiovascular diseases, osteoporosis, Alzheimerâs disease, diabetes and atherosclerosis (44). The human IL-6 gene is located on chromosome band 7p21 and consists of 5 exons and 4 introns (45). Genetic studies have identified 4 polymorphisms in the Lattanzio et al 121 promoter region of the IL-6 gene (-597G/A, -572G/C, - 373A/T, -174G/C) that have a significant effect on IL-6 expression in an in vitro system (46). A variable number of tandem repeat (VNTR) polymorphisms were found in the 3â flanking region of the IL-6 gene (C allele) (47). In humans, a polymorphism in the IL-6 promoter (A/C polymorphism at position -174) altered IL-6 gene transcription rates in vitro (48) and IL-6 levels in vivo (49). In this last paper, people carrying CC or GC genotypes are defined as C+, while those carrying GG genotypes are indicated as C-. It has been suggested that IL-6 -174G/C locus variability is capable of modulating, on the one hand, individual susceptibility to common causes of morbidity and mortality among elderly, while, on the other hand, it may play a crucial role in longevity (50). Therefore, the genetic variations of this locus of IL-6 gene are fundamental in the elderly population to better understand the intrinsic causes of the longevity. The association of these genetic variations to the possible, different immune responses is an attractive focus in elucidating the molecular mechanisms involved in immunosenescence. The genetic variations of the IL-6 -174G/C locus have been extensively studied by different groups with, however, contradictory data. BonafÃ¨ et al (51) studied IL-6 promoter genetic variability at the -174C/G locus and its effect on IL-6 levels in 700 Italians aged 60 to 110 years, including 323 centenarians. Individuals who are genetically predisposed to produce high levels of IL-6 during aging â i.e., C- men (GG genotype) â at the IL-6 -174C/G locus are disadvantaged for longevity. On the other hand, the capability of C+ individuals (CC and CG genotypes) to produce low levels of IL-6 throughout their life span appears to be beneficial for longevity, at least in men. Women have, conversely, high IL-6 serum levels later in life with respect to men, independently of -174C/G locus polymorphism (51). The inhibitory tone of estrogens on IL-6 gene expression could explain the sex-based difference (52), assuming that its long-term effects last until the extreme limits of the human life span. The major production of IL-6 in C- subjects for their whole life, including centenarians, has also been confirmed by other in vivo longitudinal studies (53). A more recent study in older and nonagenarian subjects has confirmed that IL-6 production is higher in C- carriers and that these subjects are prone to contract one of the more common age-related inflammatory pathologies such as atherosclerosis (54). Interestingly, in this last study, C- older and nonagenarian subjects also display impaired innate immune response (NK cell cytotoxicity) coupled with increased MT, zinc deficiency, and low zinc ion availability, in comparison with C+ carriers (54). A functional effect of the IL-6 -174G/C locus on MT expression and zinc-regulated genes has also been confirmed recently by targeted studies with in vitro and in vivo zinc supplementation (55, 56). These findings clearly suggest that the genetic variations of the IL-6 -174G/C locus play a key role in longevity also at a functional level, and confirm the existence of a higher number of C+ centenarians than Câ ones as previously found by BonafÃ¨ et al (51). In this context, an intriguing point is the low expression of the signal-transducing component (gp130) of the IL-6 receptor in centenarians, in comparison with the elderly, despite high circulating levels of IL-6 (57), which in turn may be inactive in centenarians (36). Therefore, inflammatory status is not so detrimental in very old age, as, in contrast, occurs in normal aging. However, the discrepancies among all of the genetic studies on the variations of the IL-6 -174G/C locus in relation to longevity in the elderly means that the subject is still unclear. Ethnic, lifestyle, and cultural differences among these populations could also play a role, as well as other undefined factors. Large-scale studies at the European level in many different ethnic populations are needed to clarify this important topic. For this reason, ZINCAGE project (www.zincage.org) has been developed, and the clarification of this point has been one of is tasks. The determination of the genetic variations of the IL-6 - 174G/C locus associated with a dietary assessment or with a comprehensive evaluation of zinc status has been reported as an useful strategy to identify older subjects who can benefit from zinc supplementation without health risks (see next paragraph).

ZINC SUPPLEMENTATION IN THE ELDERLY ON THE BASIS OF GENETIC BACKGROUND One possible cause of the discrepancies existing in the literature on the effect of zinc supplementation upon immune response and antioxidant performance in the elderly (see review in (58)) may be the choice of older subjects who effectively need zinc supplementation in strict relationship with dietary habits and inflammatory status. This fact is supported by the discovery that older subjects carrying GG genotypes (i.e., C- carriers) in the IL-6 -174G/C locus display increased IL-6 production, low intracellular zinc ion availability, and impaired innate immune responses coupled with enhanced MT (56). By contrast, older subjects carrying the GC and CC genotypes (C+ carriers) in the same IL-6 -174 locus display satisfactory intracellular zinc as well as innate immune responses. But, the more intriguing finding is that males carriers of the C+ allele are more likely to reach centenarian age than C- carriers. Therefore, older C- subjects are more likely to benefit from zinc supplementation than older C+ carriers. The distribution of the IL-6 -174 genotype is very different among the various European countries, with large differences between northern and southern European countries (58). Zinc supplementation in older C- subjects restores NK cell cytotoxicity to values present in older C+ carriers and considerably improves both zinc status, assessed by the percentage increment of granulocyte Zn (56), and stress response, assessed by the percentage increment of clusterin (CLU) expression and MT protein (58). When the genetic variations for the IL-6 polymorphism are also associated with the genetic variations of MT1A in position +647, the plasma zinc deficiency and the altered immune response are more evident (58), suggesting that the genetic variations of IL-6 and MT1A would be very useful tools for the choice of older people who effectively need zinc supplementation. These results open the hypothesis that the daily requirement for zinc might be different in elderly with a different genetic background. Such a role played by genetic background on the beneficial effects of zinc supplementation is also evident in the better control of proinflammatory cytokine and chemokine production (59), as well as in reducing the expression of genes related to inflammatory status, such as IL-1 and its receptor (55). A very intriguing point is also the beneficial effect of zinc supplementation in the cognitive performance of older individuals selected on the basis of IL-6 polymorphism (60). In any case, from a microarray analysis in vitro, zinc at a dose of 50 mM down-regulates the gene expression of proinflammatory cytokines (Fig. 1), strengthening its antiinflammatory role (56, 58).

Fig. 1 - In vitro zinc treatment (50 ÂµM) down-regulates proinflammatory cytokine expression (custom array) when refereed to the value 1 (baseline control) in human peripheral blood mononuclear cells from young adult donors. The red square represents the proinflammatory cytokines (IL-6, IL-1, and TNF pathways). Data redrawn from the Zincage Project (www.zincage.org) (61).

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BIOG 1440 Lecture Notes - Lecture 4: Metabolic Pathway, Ionic Strength, Activation Energy

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