biochemistry exam #2 content

enzymes, nucleic acids, DNA replication

Michaelis-Menten equation

v₀= v_max[S] / K_m+[S]

what is v₀?

initial velocity, slope at the start of the rxn

what is v_max?

max velocity

it’s when all the enzyme has been bound to the substrate

where the curve plateaus

equation for v_max (besides reworked Michaelis-Menten equation)

v_max=K_cat[E_t]

where E_t is the total enzyme concentration

what is k_m?

Michaelis-Menten constant

measure of substrate’s affinity for an enzyme.

on the plot, it is substrate concentration at ½ v_max

k_m equation

k_m= v_max/2 = (k_-1 + k₂₎ / k₁

what is k_cat?

turnover #, or number of substrate molecules converted to product per unit time when 100% of the enzyme active sites are bound to the substrate (SPEED of conversion)

to find on plot, find v_max and then divide by total enzyme concentration

k_cat = v_max / [E_t]

what is k_cat / k_m?

specificity constant: how efficiently enzyme converts substrate into a product at low [S]

overall describes catalytic efficiency of an enzyme

k_cat / k_m equation

k_cat / k_m = v₀ / [E_t][S]

what does a lower k_m mean?

enzyme has high affinity for binding to a substrate

enzyme reaches half of its max velocity at a low substrate concentration

what does a higher kcat/km mean?

enzyme is very efficient because it has a fast turnover rate w/ strong binding affinity.

at a low substrate concentration, what is velocity doing?

increases proportionally with the substrate concentration

at a high substrate concentration, what is velocity doing?

approaching vmax

eventually, further increases in [S] have no effect on rxn rate because there are no more free active sites on the enzyme

competitive inhibition definition

inhibitor and normal substrate have similar structure, so they compete to fit into the same active site on the enzyme. the inhibitor can directly block the substrate from binding to the enzyme.

can be overcome if [S] is high enough

what happens to Lineweaver-Burk plot if there’s competitive inhibition?

• K_m increases b/c a higher [S] is needed to reach ½ vmax

• vmax the same

• slope changes

• y-intercept the same

uncompetitive inhibition definition

inhibitor binds exclusively to ES complex and never the free enzyme. this can lock the enzyme in the ESI complex

what happens to Lineweaver-Burk plot if there’s uncompetitive inhibition?

k_m decreases

v_max decreases b/c some of the enzyme is locked in the ESI complex

slope the same

y-int the same

mixed (allostertic) inhibition definition

inhibitor reduces an enzyme’s activity by binding to an allosteric site on either the enzyme or ES complex

what happens to Lineweaver-Burk plot if there’s mixed inhibition?

km increases if inhibitor binds stronger to free enzyme. km decreases if inhibitor binds stronger to es complex

vmax decreases

pure noncompetitive inhibition definition

special case of mixed inhibition where inhibitor has equal affinity for E and ES

km no change

vmax decreases

slope changes

y-int changes

substrate binding depends on?

1. h-bonds

2. electrostatic interactions

3. hydrophobic interactions

4. van der Waals interactions

enzyme specificity depends on?

geometric and chemical complementarity b/w enzyme and substrate

cofactor

nonpreotin component needed for enzyme activity

coenzymes

organic cofactor

cosubstrate

type of organic cofactor that loosely binds to the enzyme, acting as a carries of chemical groups or electrons.

gets released from enzyme during rxn

prosthetic group

tightly/covalently bonded cofactor to enzyme

doesn’t get released from enzyme during rxn

apoenzyme

enzyme w/o its required cofactor

holoenzyme

active enzyme w/ its required cofactor

acid-base catalysis

proton transfer; lowers thre free energy of the rxn’s transition state

covalent catalysis

temporary covalent bond forms b/w the enzyme and the substrate, creating an alternate, lower-energy rxn pathway

requires a nucleophile on the enzyme

metal ion cofactor catalysis

metal ions stabilize negative chargers, properly orient substrates, activates water/nucleophiles, which all together lowers energy of transition state

proximity and orientation catalysis

brings reacting groups closer together and holds them in correct orientation and restricts rotational freedom. this lowers the energy needed to start the rxn, making it more thermodynamically favorable

electrostatic/transition state stabilization

enzymes work to stabilize developing charges in the transition states, alter amino acid pka and reorganizes water. Together, this creates electrostatic environments favorable for the rxn.

catalysis results from many weak interactions acting together

5 ways enzyme activity is regulated

1. allosteric regulation: activators/inhibitors bind to allosteric site, causing enzyme conformational change

2. feedback inhibition: end product of a metabolic pathway acts as a negative regulator, inhibiting an enzyme earlier in the pathway

3. reversible covalent modification: enzymes are activated or deactivated by the addition or removal of chemical groups

4. proteolytic cleavage: enzymes synthesized as zymogens and activated when a specific peptide bond is cleaved

5. gene expression

6. inhibitors

transition state theory

says that enzymes speed up reactions by lowering the energy needed for the reaction to occur. enzymes preferentially bind most tightly to the transition state, lowering its energy, and thus lowering the energy barrier needed to get there

key characteristics of serine proteases

• secreted by pancreas into digestive tract to break down proteins into peptide bonds and amino acids

• use a reactive serine residue as the primary nucleophile to attack peptide vbonds

• contain the catalytic triad of 3 amino acids: serine, histidine, and aspartic acid

• secreted as zymogens that are activated by proteolysis

zymogen

inactive enzyme precursor

serine proteases use which catalytic strategies?

1. acid-base

2. covalent

3. proximity/orientation

4. electrostatic/transition state stabilization

chymotrypsin cleaves…?

peptide bonds after phe, tyr, and trp

trypsin cleaves…?

peptide bonds after lys and arg

elastase cleaves…?

peptide bonds after gly, glu, val, leu, ile (small uncharged)

nucleotides are made of?

nitrogenous base, pentose sugar (deoxyribose or ribose), and one or more phosphate groups

nucleosides are made of?

nitrogenous base and pentose sugar

how are nucleotides connected to each other?

phosphodiester bond between 3’ oxygen of one sugar to the 5’ phosphate of the other nucleotide

what bond attaches the nitrogenous base to the pentose sugar in nucleotides/nucleosides?

glycosidic bond via the sugar’s C1’ to a nitrogen atom of the base

purines attach via N9

pyrimidines attach via N11

which DNA polymerase synthesizes the leading strand? (prokaryotes and eukaryotes)

prokaryotes: DNA polymerase III

eukaryotes: DNA polymerase ε (epsilon)

which DNA polymerase synthesizes the lagging strand? (prokaryotes and eukaryotes)

prokaryotes: DNA polymerase III

eukaryotes: DNA polymerase δ (delta)

which DNA polymerase replaces the RNA strand? (prokaryotes and eukaryotes)

prokaryotes: DNA polymerase I

eukaryotes: DNA polymerase δ

processivity

refers to an enzyme’s ability to catalyze consecutive chemical reactions, specifically DNA polymerase in adding nucleotides to a growing DNA strand w/o releasing its DNA template.

repetitive DNA polymerases need high processivity to copy long stretches of DNA quickly and efficiently.

how is processivity measured?

average number of nucleotides a DNA polymerase incorporates in a single binding event before dissociating from the template

topoisomerase general function

relieves torsional stress ahead of the fork

helicase general function

unwinds DNA double helix

SSBs general function

stabilize separated DNA strands and keeps them separated so that replication can occur

rho factor general function

terms for template strand

antisense

noncoding

terms for nontemplate strand

sense strand

coding strand

how are Okazaki fragments joined together?

1. primase repeatedly makes RNA primers on the lagging strand

2. DNA polymerase extends each primer to form an Okazaki fragment

3. DNA polymerase removes RNA primers and replaces them w/ DNA

4. DNA ligase seals remaining nicks in the sugar-phosphate backbone

explain DNA replication process in bacteria

explain DNA replication process in eukaryotes

initiation

1. at multiple origins of replication, helicase breaks the bonds holding the two DNA strands together, forming a replication fork. topoisomerase reduces torsional stress ahead of the fork.

elongation

• primase lays down RNA primers to give DNA polymerase the 3’-OH group it needs to latch on to.

• DNA polymerase builds the new DNA strand from the template. It READS the strand in the 3’ to 5’ direction. It BUILDS the strand in the 5’ to the 3’ direction.

• leading strand is built continuously toward the replication fork.

• lagging strand is built discontinuously in the direction away from the replication fork.

termination

• RNA primers and replaced with DNA bases

• DNA ligase seals all the nicks in the Okazaki fragments.

exons

nucleotide sequence that codes for amino acids to build proteins

stays in mature mRNA after it gets processed

introns

nucleotides sequence that’s noncoding

removed from pre-mRNA

mRNA function

carries nucleotide sequence that specifies a polypeptide

tRNA function

delivers amino acids to the ribosome during translation

rRNA function

forms structural and catalysis of ribosomes

What are the steps of PCR?

1. denaturation: heat separates DNA strands

2. annealing: primers bind complementary sequence

3. extension: DNA polymerase extends the primer

why are 2 primers used in PCR?

to make sure both strands of the double-stranded DNA are amplified. if only one primer is used, only one strand will be amplified.