Biochem Exam 3

What are the 5 nitrogenous bases?

Adenine, Cytosine, Guanine, Thymine, Uracil

The purines are _ and _. They are bigger than pyrimidines because of the 5 carbon group. Purines have _ nitrogens vs pyrimidines have _ nitrogens

adenine and guanine; 4 and 2

The pyrimidines are _,_, and _. These are more planar than purine.

Cytosine, Uracil, Thymine

All of the pyrimidine and purine bases absorb _._. light so the concentration of DNA or RNA in a sample can be determined by measuring the \__ absorbance.

UV; UV

Probably don't need to know:
Keto-enol tautomeric shift:
In the Enol form, the ring nitrogens can serve as H-bond donors, and the keto oxygens serve as H-bond acceptors, when interacting with other molecules.

cool

RNA and DNA contain _ _ in addition to a purine or pyrimidine nitrogenous base as part of their structure:. RNA contains a _ molecule and DNA contains a _-_ molecule (2'-deoxy-ribose)

ribose sugars; ribose; deoxy-ribose

The complete structure of a _ _ plus a _ _ ( _ bond) is called a Nucleoside

nitrogenous base; ribose sugar; glycosidic

The Glycosidic bonds in nucleosides are always in the _ form

beta

Nucleosides are named by adding the suffix "-_" to the pyrimidine or "-_" to the purine name.

idine; osine

What is the number of the carbon that determines the "deoxy" designation of a nucleoside?

2

A nucleotide \= _ + _ + _

(can be purine or pyrimidine) nitrogenous base; sugar (ribose or deoxyribose); phosphate group

Match the molecule to its primary purpose:
\___\= Central to energy metabolism
\___\=Drives protein synthesis
\___\= Drives lipid synthesis
\___\= Drives carbohydrate metabolism

ATP, GTP, CTP, UTP

What kind of reaction adds a phosphate group to a nucleotide? What kind of reaction removes a phosphate group from a nucleotide?

Phosphorylation; Hydrolyzation

Transfer RNA transports _ _ to site of _ synthesis

amino acids; protein

Ribosomal RNA combines with _ to form _, the site of _ synthesis

proteins; ribosomes; protein

Messenger RNA directs _ _ _ of proteins

amino acid sequence

Small nuclear RNA processes _ _ to its mature form in _

initial mRNA; eukaryotes

Small interfering RNA affects _ expression; used by scientists to knock out a gene being studied

gene

Micro RNA affects _ expression; important in _ and _ development

gene; growth and development

Which of the following is true about phosphodiester linkage? 5'-phosphate group of one nucleotide unit is joined to the 3'-hydroxyl group of the next nucleotide.
(Strands run in opposite 5' to 3' directions!)

Fair

What type of reaction is used to create the phosphodiester bonds in nucleic acids?

Condensation

Double helix pairs:
Guanine (G) is always paired with what?
Thymine (T) is always paired with what?
For RNA Uracil (U) is paired with what?

Cytosine (C); Adenine (A); Adenine (A)

Double helix bonds nucleotides have _ bonding between the nucleotides. Just know that DNA helix has a major groove and a minor groove.

hydrogen

Very important:
G-C base pairs are stabilized by _ hydrogen bonds, whereas A-T base pairs are stabilized by _. This means that the G-C interaction is _ than the A-T interaction

3; 2; stronger

Exonucleases start at the end of a _ (either the 3' or 5' end, depending on their specificity) and sequentially _ the phosphodiester bond in the polynucleotide.
Endonucleases _ at an _ site within the polynucleotide.
(Nucleases can cleave on either side of the phosphodiester bond.)

polynucleotide; hydrolyze; cleave; internal

Exonuclease type a cleaves at the _ end and type b cleaves at the _ end of the phosphodiester bond.
(Nucleases can be single or double stranded)

3'; 5'

_ _ are those that cleave DNA based on specific sequence sites. This allows researchers to cleave DNA for processes like fingerprinting, cloning etc. There are 3 types of these.

Restriction Endonucleases

Type 1 restriction endonuclease
-Requires \___ for hydrolysis
-Is able to _ DNA at _ locations
-Cuts at _ sites

ATP; methylate; specific; random

Type 2 restriction endonuclease
-Does not require \___ for hydrolysis
-Does not _ DNA
-Cuts at _ sites

ATP; methylate; specific

Type 3 restriction endonuclease
-Requires \___ for hydrolysis
-Is able to _ DNA at _ locations
-Cuts at _ nucleotide sequences

ATP; methylate; specific

The primary structure _ are linked together by phosphodiester linkages to form a _ strand. The _ _ links the 3' and 5' oxygens of _ sugars.

nucleosides; single; phosphate esters; 2

The DNA secondary structure is a _ helix formed by 2 _-_ DNA strands bind together by _ bonding between bases on _ strands

double; anti-parallel; hydrogen; opposite

The structure of DNA in a chromosome is considered a _ structure

tertiary

_ _ distorts the DNA double helix interfering with DNA replication, transcription, and repair.

Intercalating agents

Supercoiling occurs when there are rotation issues with the DNA strands or too much strain on the strands during the rotations. Supercoiling is important for \___ _ within all cells. Which is important because the DNA is too big to be packaged so coiling it using supercoiling makes it easier for DNA replication. The _ _ serves two purposes:
1. Yield a more compact shape
2. Can result in unwinding of DNA helix (aids in replication)

DNA packaging; superhelical turns

Two enzymes work to maintain supercoiling in DNA. _ relax supercoiled DNA and \___ _ introduces supercoiling.

Topoisomerases; DNA gyrase

Negative supercoiling - _-handed - strands are _.
Positive supercoiling - _-handed - strands are overwound
( ALSO Know which supercoiling is more common in the cell )

left; underwound
right; overwound
(Negative supercoiling is more common)

_ _ (L) - This is the basic parameter used to characterize supercoiling. It is the number of times the two DNA strands are intertwined.

Linking Number

The Linking number can also be equated to parameters called the Twist (T) and the Writhe (W). The twist is the number of helical turns and the writhe is the number of supercoils. The formula is what?:

L \= T + W

B-DNA is the normal DNA we have and it is longer, skinnier, and hydrated. The helical rotation is to the right.

A-DNA is the normal form for (double stranded) RNA and it is fatter, wider, and dehydrated. The helical location is to the right.

Z-DNA is long, skinny, and occurs in stretches of alternating purine-pyridine base pairs. The helical location is to the left.

The DNA duplex is held together by a combination of _ bonds between base pairs and the _ _ _ forces associated with base stacking interactions along the helix. These interactions can be disrupted using pH, temperature or ionic strength.

hydrogen; van der Waals

Upon addition of heat, a phase transition will occur at a characteristic "melting" temperature (TM). At this temperature, the DNA duplex will "melt" yielding _ and _ single strands of DNA. The TM is related to the proportion of the _ _ in the DNA.

separated; denatured; different bases

Renaturation of DNA duplex means the two strands have to return to the correct _ _ order. The amount of time required for this renaturation to occur will be directly related to the _ of the \___ sequence.

base pairing; complexity; DNA

_:
-Represents the major genes and is involved in transcription
-Chromosome material which doesn't stain strongly except during cell division
-Comprises the most active portion of the genome within the cell
-lighter staining is due the less compact structure of euchromatin
-also participates in the active transcription of DNA to mRNA products

Euchromatin

_:
-chromosome material of different density from normal (usually greater), in which the activity of the genes is modified or suppressed
-usually localized to the periphery of the nucleus and it stains intensely, indicating tighter packing
-mainly consists of genetically inactive satellite sequences and many genes are repressed to various extends although some cannon be expressed in euchromatin at all

Heterochromatin

Proteins in chromatin are the _ which are relatively small, positively charged proteins of 5 major types.

Histones

What are the 5 major types of histones?

H1, H2A, H2B, H3, H4

The 2 most abundant amino acids found in histones are _ and _, positively charged amino acids that give them a net positive charge. The positive charges attract the negative charges on the phosphates of DNA and holds the DNA in contact with the histones.

Arginine and lysine

_ _ (\____):
Carries the message encoded in DNA to locations within the cell where protein synthesis takes place
-_ is synthesized during the process of transcription
-A enzyme (RNA polymerase) copies the information from a DNA template into a complementary RNA molecule
-The information in the _ is then translated into a corresponding protein sequence by the protein synthesizing machinery within a ribosome

Messenger RNA (mRNA)

_ _ (\____)
-the function is not fully understood but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation.

Ribosomal RNA (rRNA)

_ _ (\____)
are carrier molecules for amino acids during protein synthesis. They transfer amino acids from cytoplasm to the protein synthesizing machinery. (Smallest of the RNA molecules and are EASILY soluble).

In translation, each of the 20 amino acids has at least one unique _ whose job is to bind to the amino acid and insert it at the correct spot in the synthesis of a protein from mRNA in the ribosome. The 3' end has the sequence CCA. The 3'-OH of the A base forms an acyl ester bond to the amino acid it carries (the entire complex being known as an amino-acyl-tRNA)

Transfer RNA (tRNA)

_ _ \___ (\_____)
-these are involved in the process of splicing (intron removal) of primary transcript to form mature mRNA. The _ form complexes with proteins to form Ribonucleoprotein particles called snRNPs.

Small Nuclear RNAs (snRNA)

RNA vs DNA major differences:
-\___ has OH group at 2' position, whereas, \___ has only a hydrogen
-_ contains Thymine, _ has Uracil instead

RNA; DNA; DNA; RNA

The restrictions RNA presents are:
-RNA can't form a duplex like B- DNA. This is because of steric hindrance of the 2'-OH
-RNA can form a duplex more like A-form DNA, with 11 bp/turn and the base aromatic rings tilted away from a perpendicular angle to the axis of the duplex
-One of the fundamental types of secondary structure for RNA is the "stem-loop" structure. (Eg. tRNA)
-More complex tertiary structures are built up from the interactions between two or more stem-loop structures

Cool shit

what are the three distinctive features enzymes are characterized by?

catalytic power, specificity, and regulation

catalytic power

an enzymes ability to increase rxn rates up to 10^14 times in comparison w/ the non-enzyme catalyzed rate

specificty

refers to the fact that enzymes are highly specific to the reaction they catalyze

regulation

enzymes may be regulated through the interaction w/ specific inhibitor or activator molecules

What are the 6 classes of enzymes?

-oxidoreductase
-transferases
-hydrolases
-lysases
-isomerases
-ligases

Oxidoreductases

catalyze oxidation-reduction reactions

transferases

transfer functional groups

hydrolases

hydrolysis reactions

lysases

addition of double bonds

isomerases

catalyze the rearrangement of bonds within a molecule
-isomerization reactions

ligases

formation of bonds w/ ATP cleavage

What is the importance of coenzymes and cofactors?

-many enzymes require an addition non-protein molecule to be active
-enzymes cannot do redox rxns and carbon-group tranfers
-some vitamins are cofactros

haloenzymes

enzymes with their bound cofactor

apoenzyme

enzymes without their cofactors

Prothestic groups vs cofactor

prosthetic group tightly binds with the enzyme to assist enzyme while coenzyme loosely binds with an enzyme to support its catalytic function

How does the concentration of substrate influence the rate an enzyme works?

influences the speed of the rxn up to a point

How does the concentration of enzyme influence the rate an enzyme works?

more enzymes, more product/activity

How does temperature influence the rate an enzyme works?

enzymes only work in a region of temperature, but as temp increases (up to a certain max temp), the speed of the rxn increases

How does the presence of an inhibitor influence the rate an enzyme works?

stops rxn

How does pH influence the rate an enzyme works?

depending on pH, can increases or decrease, or stop activity

What are the forces that attribute to substrate specificity?

the non-covalent bonds are forces are maximized to bind w/ substrate specificity
-van der Waals forces
-electrostatic bonds (ionic interactions)
-hydrogen bonding

Why are these forces important?

weak forces allow for reversible interactions

What does it mean when an enzyme is saturated?

at high substrate (S) concentrations, the enzyme may become saturated

What happens at saturated conditions of an enzyme?

-under saturated conditions, the enzyme is working as fast as it can, and the rxn willl not increase even if more reactant is added
This is called Vmax

What order kinetics do enzymes follow when substrate concentrations are high?

zero order kinetics
there is no increase in the rate of the rxn when more substrate is added because the enzyme is already working as fast as it can

Vmax

maximum initial velocity or rate of an enzyme-catalysed reaction.

Km

Substrate concentration at 1/2 Vmax

kcat

turnover number (molecules catalyzed per second in optimal conditions)Vmax / [E]

Catalytic Effeciency

Kcat/Km

diffusion limit

-The upper limit for kcat/Km
-the rate at which E and S diffuse together

What is the relationship between Km and enzyme affinity?

The value of KM is inversely related to the affinity of the enzyme for its substrate.
-High values of KM correspond to low enzyme affinity for substrate (it takes more substrate to get to Vmax).
-Low KM values for an enzyme correspond to high affinity for substrate.

Lineweaver-Burk equation

1/v \= (Km/Vmax)(1/[S]) + 1/Vmax

What is an inhibitor?

inhibitors are molecules or compounds that interact w/ an enzyme and decrease its affinity

reversible inhibitors

have affinity for an enzyme via non-covalent interactions

irreversible inhibitors

interact via stable, covalent interactions

What are the reversible inhibitor types?

competitive inhibitors, noncompetitive inhibitors and uncompetitive inhibitors

competitive inhibitors

compete with substrate for active site
-this form of inhibition can be overcome by increasing the substrate concentration to outcompete the inhibitors

How do competitive inhibitors work?

-look like the substrate
-bind to active site
-increase Km
-constant Vmax

noncompetitive inhibitor

reversible inhibitors react with both E and ES b/c the inihibtor binds at a site other than the active site
-this type of inhibition cannot be overcome by increasing the substrate concentration

How do noncompetitive inhibitors work?

-don't look like substrate
-bind to a site other than active site
-the effects on kinetics is as if the enzyme were less active (Vmax is reduced)
-the affinity for the substrate is unaffected (Km remains the same) since the substrate binding site is not occupied by the noncompetitive inhibitor

uncompetitive inhibitors

- bind directly to the enzyme substrate complex, but not to the free enzyme
- bind causes a conformational change at the active site that renders the enzyme inactive
- both max and Km are changed and they both decrease
- slope of the line is parallel with the slope of the uninhibited reaction

How do uncompetitive inhibitors work?

-bind at alt. site than active site-Km decreases-Vmax lowered

irreversible inhibition

this type of inhibition renders the enzyme "dead" and with no change of recovery
-reduction in Vmax
-Km is unaffected
-can bind either at active site or inactive site

How can inhibitors be removed?

while reversible inhibitors can be removed by dialysis (i.e. a diffusion processs), irreversible inhibitors cannot be removed
-this difference provides an experimental method by which the type of inhibitor can be determined

Single Displacement/Replacement

E + A + B -\> AEB -\> PEQ -\> E + P + Q
E\= enzyme
A\= substrate one
B\= substrate two
P\= product one
Q\= product two

What are the two types of single/sequential displacement reactions?

random and ordered