C+M Exam 1

Most mutations are _, some are _ and some can contribute to _

silent, harmful, increased ability of the organism to survive and adapt

all diseases are__

disturbances at the cellular level

as ions become hydrated, the attractive force between them is _, and the charged species dissolves in the water

reduced

hydrophobic effect

nonpolar substances are EXCLUDED from the solvation netweork

ordered water cage

is around each nonpolar molecule, limits water molecules from interacting with other water molecules

lowest energy state in hydrophobic effect

has maximum water-water hydrogen bonding and minimal exposure of hydrophobic surfaces

gene

region of DNA that can be expressed to produce a final functional product that is either a protein or RNA molecule

proteins add___

functionality

functionality

puts cell’s genetic info into ACTION

functions of proteins

1. structural
2. movement
3. defense
4. regulation
5. transport
6. catalysis

each protein normally folds into a __ stable conformation

single stable

general formula of an amino acid

1. amino group
2. carboxyl group
3. side chain group (R)

how many side chains are there?

20

At pH __ both the amino and carboxyl group are ionized

7

nonpolar side chains

some can be remembered with “glaciers in alaska valiently locate isolated prowlers”


1. gycline (G)
2. alanine (A)
3. valine (V)
4. leucine (L)
5. isoleucine (I)
6. proline (P)

Then there are


1. Phenylalanine (F)
2. Methionine (M)
3. Tryptophan (W)
4. Cysteine (C)

Uncharged polar amino acids

Asparagus Never Gives Quacks Seriously Sufficient To Thoroughly Tyre You.


1. Asparagine (N)
2. Glutamine (Q)
3. Serine (S)
4. Threonine (T)
5. Tyrosine (Y)

Positive (basic) polar amino acids

Can be remembered with “basically, His Lost kid Always returned”


1. histidine (H)
2. lysine (K)
3. Arginine (R)

Negative (acidic) polar amino acids

As peter digested the glue, his stomach became acidic


1. Aspartic acid (D)
2. Glutamic Acid (E)

chiral carbons exist when the alpha carbon is attached to __ different groups

4

molecules with chiral carbons exist as __

stereoisomers

L-amino acids form a ___ helix

right handed

(T/F) Enzymes are chiral

T

Protein structure dictates __

function

peptide bonds

amide linkage that joins the amino acids

primary level of protein organization

linear amino acid sequence

secondary level of protein organization

basic folding units (alpha helices and beta pleated sheets)

tertiary structure of protein organization

3-D structure of a folded protein - non-covalent interactions of side groups

quaternary structure of protein organization

complete 3D structure of proteins that contain more than one polypeptide chain

Sequences are listed from _ terminus to _ terminus

N to C

a protein’s 3D structure is determined by the

order of amino acids in its chains

backbone of the polypeptide is composed of

everything but the R group

alpha helix

1. occurs when there is H bonding between N-H and C=O groups in polypeptide backbone
2. R groups project AWAY from helix
3. can be amphipathic - both polar and nonpolar (happens when 1 side is polar and the other side is nonpolar)

coiled coil

* supersecondary structure from multiple alpha helixes wrapping around one another
* 2+ helices have most of their nonpolar side chains on 1 side, twist around each other with side chains facing inwards (hydrophobic effect)

beta pleated sheet

* forms when 2+ polypeptide side chains line up side by side
* each beta strand is fully extended and stabilized by H bonding between N-H and carbonyl groups
* Can be parallel or antiparallel

example of beta pleated sheet strength in real life

spider silk

protein domain

* a substructure produced by any contiguous part of a polypeptide that can fold independently into a stable, compact structure
* typically consist of 50-350 amino acids
* each domain conders a specific function to the protein
* even a tertiary structure can have domains

globular proteins vs. fibrous proteins

globular - dynamic and soluble in water

fibrous - insoluble in water, structural, elongated

non-covalent interactions that stabilize tertiary structure

1. van der waals attractions: weak bond froms at a close range between nonpolar atoms
2. hydrogen bonding (weak on its own but can have multiple H-bond that make it strong) - hydrogen atoms and usually O or N
3. Electrostatic attractions - salt bridges (attraction between ionic groups of opposite charge)

many inheritable genetic disorders are due to ___

deficiency/absence/excess activity of one or more enzymes

how can measurements of enzyme activity be useful?

diagnostics

many drugs exert their biological effects through interactions with __

enzymes

enzymes __ activation energy

lower

(T/F) Enzymes alter the standard free energy of a reaction

FALSE

phosphotases

catalyze hydrolytic removal of a phosphate group from a molecule (remove phosphate groups)

kinases

catalyze the addition of a phosphate group to a molecule (add phosphate groups)

ATPase

hydrolyze ATP. energy-harnessing ATPase activity as part of their function (nucleotide regulation)

GTPase

hydrolyze GTP, help regulate cell processes (molecular switch - either active or inactive) (nucleotide regulation)

protease

break down proteins by hydrolyzing peptide bonds between amino acids

(T/F) the active site uses only one amino acid side chain to coordinate making/breaking bonds with substrates

FALSE - SEVERAL AMINO ACID SIDE CHAINS INVOLVED

What is the 3D structure of the reactive center formed by?

folded domains of the protein

enzymes are ___ for their substrates

highly specific

(T/F) Enzymes are consumed

FALSE - can be used over and over again

substrate

reactant that binds to the active site of a protein

induced fit model

the shape of the active sit becomes truly complementary only after the substrate begins binding to the enzyme

proteins can have slight conformational changes

gives them flexibility - is how induced-fit model can happen

ligand induced conformational changes

increased or decreased binding affinity of an enzyme from a DIFFERENT MOLECULE

Ex. After glucose binds to hexokinase, there is a 50 fold increase in the affinity of the enzyme for ATP

general strategies of enzyme catalysis

1. align/position reactants
2. induce charge state
3. deform reactants

algin/position reactants

* fewer conformations to explore
* increase efficiency

induce charge state

amino acids in active site interact with reactants via charge, polarity

deform reactants

* enzyme strains substrate
* forcing a transition state, favor reaction

lysozyme

* catalyzes the cutting of polysaccharide (sugar) chains in cell walls of bacteria
* catalyzes a hydrolysis reaction: adds water to bond between two adjacent sugar groups in sugar chain to cause bond breakage, reaction is favorable

lysozyme reaction pathway

1. sugar D is forced into a strained conformation
2. Gly35 serves as an acid to donate a H+ to sugar E
3. Asp52 attacks C1 of Sugar D, TRANSIENT COVALENT BONDS form between Asp and Sugar D
4. Hydrolysis of glycosidic bond (sugar-sugar bond)
5. Glu35 polarizes the water molecule, oxygen attacks C1
6. COVALENT BOND BROKEN between Asp and C1 (sugar D)
7. Lysozyme returned to initial state (EP formation)

review the slide for this - definitely on exam

Enzyme characteristics

1. enzymes are catalysts - lower activation energy, promote the transition state
2. Enzymes are highly specific to reaction they catalyze
3. Enzymes work in moderate temperature
4. Enzymes are NOT consumed in reactions
5. Enzymes CAN be regulated

types of protein regulation

1. allosteric regulation
2. irreversible inhibition
3. post translational modifications
4. proteolytic processing


1. nucleotide regulation

Most proteins are allosteric, which means that ___

binding at one of the sires causes a shift from one folded shape to a slightly different folded shape

allosteric inhibition

binding of one molecule inhibits binding of a substrate (can’t both be binded)

allosteric activation

binding of one molecule allows for the binding of a substrate (both want the same conformation)

feedback inhibition (negative feedback)

product produced late in the reaction pathway inhibits an enzyme that acts earlier in the pathway

Example of allosteric activation

ADP → ATP

ADP is an indicator of __

low energy status in the cell

cooperative allosteric transition

multiple binding sites where binding at one site increases the affinity for binding in the remaining sites (4 oxygen can bind to one hemoglobin)

example of irreversible inhibitor

aspirin

reversible phosphorylation/dephosphorylation

* can activate OR inactive an enzyme
* can mask OR create a binding site
* Ex. cyclin-Cdk complexes of cell-cycle control system

proteolytic processing

some proteins/enzymes are made in inactive forms and then become active after proteolytic processing and cleavage of the precursors

Ex. insulin production

ATP

energy currency of the cell

enzymes that couple energy transduction to mechanical work

nucleotide binding and hydrolysis function in a variety of enzymes to perform work

different classes of mechanical enzymes

1. membrane transporters
2. molecular motors/machines (ATPases)

GTPases function as molecular clocks

Ras Protein (GTPase) plays a role in cell signaling

GTPase regulation

* GTPase is an indirect type of activation
* GAP - GTPase activating protein - does NOT activate a protein but it hydrolyzes GTP to turn off GTPase
* GEF - guanine exchange factor - promotes dissociation of GDP
* GTP bound = active
* GDP bound = inactive

cell fractionation

* separating cells into their component fractions
* done through osmotic shock, ultrasound vibration, forced through a small orifice, blender
* plasma membrane and ER membrane retain biochemical properties
* most organelles are left intact

homogenate (or extract)

suspension of cells, contains organelles with a distinct size, charge, and density

how do you purify mitochondria from cell homogenate?

centrifuge at a speed LOWER than what is required to pellet mitochondria

supernatant

poured into a new tube and centrifuged at a rate to cause mitochondria to pellet

salting out

* using salt to purify proteins through water interactions
* increases protein-protein interactions
* every protein has a characteristic salting out point
* salt can be denaturing so which salt you choose matters

chromatography

* mixture of proteins in solution pass through a column containing a porous solid matrix

what is the mobile phase in chomatography?

mixture of proteins

what is the stationary phase in chromatography?

the column and everything in it (resin beads, porous solid matrix,..)

how do proteins slow down in chromatography?

based on interactions with the matrix, separated as they flow through the column

ion-exchange chromatography

proteins are separated according to chargei

isoelectric point (pI)

pH when molecule has no charge

cation exchange resins

bind to positive chargesa

anion exchange resins

bind to negative charges

elution

process of extracting a substance that is absorbed by another by washing it with a solvent

pH affects the net charge of a protein

* in solutions more basic than the pI, the amino group donates a proton
* in solutions more acidic than the pI, the carboxyl group accepts a proton

Gel-filtration chromatography

aka “size exclusion”

* molecules that are small will linger in porous beads
* larger molecules elute faster

affinity chromatography

* uses non-covalent bonding affinity between protein and ligand


* ligand is covalently bound to insoluble matrix
* Ex. bound substrate (affinity for enzyme), bound antibody (affinity for specific protein)

SDS-PAGE

* proteins usually have a net charge
* electric field is applied to solution with protein molecules
* protein migrates at a rate that depends on its net charge, size, and shape

metabolic flexibility

quick changes in concentrations of key regulatory enzymes, hormones, receptors, etc.

chaperones function

1. assist
2. rescue
3. protect