biochem quiz 4 (copy)

pH optimum

the pH that results in the maximal activity of an enzyme

step 1 acid-base catalysis: chymotrypsin

a group can take on a H+ at a low pH when H+ from the solution diffuses into the active site. if the group already has a H+, catalysis wont happen

step 2 acid-base catalysis: chymotrypsin

a group can give up an H+ at a high pH because some base from the solution can diffuse into the active site. if the H+ was already given up, catalysis won't happen

what causes differences in the pH optimum?

amino acid sequence, structure, how they catalyze

pH optimum graph

up - take on a proton (increase in enzyme activity)
down- release a proton (decrease in enzyme activity)

serine proteases

hydrolyze bonds by using water to break them into 2 pieces

chymotrypsin

hydrolyzes peptide bonds adjacent to aromatic acids (specific) with Asp 102, His 57, and Ser 195

difference between catalysis of enzymes and non enzymes

non enzymes are not specific but enzymes are specific

what type of reaction is the ser protease mechanism?

ping pong double displacement

step 1 of the two step reaction process

-OH Ser side chain of the enzyme covalently binds to part of the substrate producing the leaving group (XH) and the acyl-enzyme

step 2 of the two step reaction process

h2o displaced the leaving group (XH) and the enzyme binds to the substrate through deacylation

convergent evolution

2 things do not have a common ancestor, but have evolved similar in function

convergent evolution in chymotrypsin and substilisin

they both cleave peptide bonds in other proteins and use Ser, Asp, and His.
His is the closest to the N terminal (lowest number)
Ser is closest to the C terminal (highest number)

site directed mutagensis

generate a polypeptide chain that is identical except Ser-221 is replaced with Ala

findings of site directed mutagensis

the Ala replacement had a lower kcat (ES = E + P) which shows the Ser-221 is important
the Km is unchanged which means the Ala is only good for E + S = ES, not for ES = E + P

enzyme regulation: maintenance of ordered state

thermodynamically unfavorable because the lower change in entropy, but some enzymes increase the change in entropy and make it favorable

enzyme regulation: conservation of energy

enzymes catalyze unfavorable reactions and use energy and if we don't need them, they are stopped

enzyme regulation: responsiveness to environmental changes

warm vs. cold has different internal changes for each

genetic control

change in regulation of transcription and translation = change in the amount of proteins produced

covalent modification

after enzyme is made in translation, another enzyme uses it as a substrate and covalently changes the polypeptide chain. PHOSPHORYLATION

allosteric regulation

something binds to the enzyme non-covalently and changes the ability to bind and catalyze

compartmentalization

enzyme is one part of the cell and substrate is in the another and regulation moves them to the same part

kinases

group of enzymes that transfer phosphate between molecules and use ATP as the phosphate donor

phosphatases

unphosphorylates an amino acid by dding water

phosphorylation

the -OH of a protein turns ATP to ADP and a phosphate is added

net result of phosphorylation

ATP hydrolysis

what process are proteases involved in?

clotting because they are active when bleeding and inactive when not bleeding (improper: stroke)

chymotrysinogen

inactive form of chymotrypsin which prevents degrading of proteins nearby and only secreted when needed

trypsin

breaks peptide bond in chymotrysinogen and makes pi-chymotrypsin which is more active

alpha chymotrypsin

fully active version of chymotrypsin

types of covalent modification

phosphorylation, methylation, acetylation, and nucleotidylation

proenzymes/zymogens

protein made as an inactive enzyme (ex: chymotrypsinogen)

what do covalent modifications in enzyme structure cause?

changes in function

serpins

proteins that inhibit Ser proteases through covalent interactions

how do serpins inhibit Ser proteases

protease catalyzes the first part of the reaction to become the acyl-enzyme intermediate and the serpin undergoes a conformational change that forces the protease to undergo a conformational change

trypsin and serpins

after the cut, the serpin conformationally changes and the loop becomes a beta sheet and pulls our of trypsin's active site so it cannot catalyze the second step of the reaction and release Ser from the serpin

proteases and drug targets

regulate blood vessel constriction, so inhibition allows relaxation which causes a drop in blood pressure

HIV has a protease needed for replication so stopping the replication prevents HIV from turning to AIDS

COVID has a protease

the anabolic process in glycogen synthesis

the glycogen is made from glucose monomers

steps of glycogen synthesis

activate the glucose, add the activated molecule to the glycogen chain, branches are created through isomerization

glycogensis

the synthesis of glycogen when glucose is stored (after a meal because high concentration of glucose)

synthesis of glucose-1-phosphate

reaction one of glycogenesis (preparatory); made from G6P with the phosphoglucomutase

synthesis of UDP-glucose

reaction two of glycogenesis (preparatory); made from G1P by the UDP-glucose phosphorylase

what energy source is used to make G6P and G1P?

ATP

what energy source is used to make UDP-glucose?

UTP and PPi is made and gives more energy because it is hydrolyzed

where does the energy of ATP come from?

phosphates

synthesis of glycogen from UDP-glucose

reaction three of glycogenesis (chain elongation and isomerization); requires glycogen synthase for growth and amylo-alpha(1,4-->1,6) glucosyl transferase for alpha(1,6) linkages for branching

why does UDP release energy?

beta-phosphate has more resonance in products than reactants and a proton is generated

what is happening here?

glycogen is being formed from UDP-glucose

what is happening here?

addition of branching enzyme forms branching in the glycogen

glycogenolysis

glycogen degradation

reaction 1 of glycogenolysis

removal of glucose from nonreducing ends (glycogen phosphorylase) within 4 glucose of branch point

reaction 2 of glycogenolysis

transfer 3 remaining glucoses to another location on the glycogen molecule via the debranching enzyme; the glycogen phosphorylase cannot act on glucoses 4 away from branch point so they have to be moved

reaction 3 of glycogenolysis

last glucose unit is removed from the glycogen via hydrolysis with the debranching enzyme and the glucose can end up in the bloodstream or glycolysis

glycogen phosphorylase

work on different part of the molecule because of branching which allows for faster G1P production and makes the release of glucose from glycogen more efficient

draw glycogenesis

draw glycogenolysis

glucagon

activates glycogenolysis

insulin

inhibits glycogenolysis and activates glycogenesis

epinephrine

inhibits glycogenesis and activates glycogenolysis

futile cycle

when glycogenesis and glycogenolysis occur at the same time

protein kinase

enzyme that uses ATP to add phosphate to a protein and regulates the glycogen phosphorylase via the cascase

active form of glycogen phosphorylase

phosphorylated

active form of glycogen synthase

unphosphorylated

draw the glycogen complex

t form of glycogen phosphorylase

unphosphorylated and minimizes glycogen bonding

r form of glycogen phosphorylase

phosphorylated and maximizes glycogen bonding

muscle cells negative allosteric regulators

ATP and G6P

muscle cells positive allosteric regulators

AMP

liver cells negative allosteric regulators

glucose

liver cells

doesn't cycle through ATP quickly and maintain blood sugar levels, so it is responsive to [glucose]

muscle cells

store glucose for themselves and degrade glycogen when running low on ATP

feedback inhibition

due to regulation of a direct product or a product further down stream of the enzyme (G6P and hexokinase)

draw feedback inhibition chart

glycogen phosphorylase active site in the r state

lys and arg in the active site allow for higher affinity for phosphate and orient themselves for acid base catalysis (decreased Km)

glycogen phosphorylase active site in the t state

loop acts as a lid and covers the binding site minimizing the glycogen binding

insulin receptor

in the membrane and sends a signal that there is insulin outside of the cell

bound insulin receptor

signal is transmitted into the cell through phosphorylating proteins and a kinase cascade occurs resulting in the glycogen phosphorylase being phosphorylated and becoming inactive

signal transduction

communication of information in biological systems

parts of signal transduction in insulin

insulin, insulin receptor, and protein kinase cascade

phosphoglucomutase

converts between G1P and G6P through isomerization

important fact about phosphoglucomutase

it does not move the phosphate from the molecule, it transfers the phosphate back to itself

which enters metabolic pathways: G1P or G6P

G6P

glycolysis stage 1

energy investment (uses ATP)

glycolysis step 1 stage 1

glucose is phosphorylated to G6P which is important because it keeps it from diffusing out of the membrane which is important because energy is put into making and taking it up (ATP USED)

draw phosphoglucomutase mechanism

glycolysis step 2 stage 1

G6P is isomerized to F6P because a free -OH is needed

glycolysis step 3 stage 1

for symmetry another phosphate is added to an -OH of F6P forming F1,6BP which is a thermodynamic driving for (ATP USED)

glycolysis step 4 stage 1

F1,6BP is cleaved in to GAP and DHAP (which is later turned into GAP)

glycolysis stage 2

the energy production phase (2 rows)

glycolysis step 1 stage 2

GAP has a thermodynamically favorable oxidation and unfavorable phosphorylation to form G1,3,BP

glycolysis step 2 stage 2

high energy molecule that can phosphorylate ADP (ATP FORMED) and forms glycerate 3 phosphate

glycolysis step 3 stage 2

isomerization occurs to form glycerate 2 phosphate

glycolysis step 4 stage 2

dehydration occurs and phosphoenolpyruvate is formed

glycolysis step 5 stage 2

high energy molecule that can phosphorylate ADP (ATP FORMED) and forms pyruvate

net ATP of glycolysis

2 ATP (2 in energy investment and 4 in energy production)

hexokinase

catalyzes conversion of glucose and ATP into G6P, ADP, and a proton

what is the first reaction of glycolysis?

hexokinase

draw glycolysis mechanism

draw hexokinase mechanism

what are the 2 types of catalysis in the ser protease mechanisms?

acid-base and covalent