Exam #3 Content

Question 1

Know all the sugars

Answer 1

-

Question 2

NH2 changed to CH3, what effect would this have on pyruvate decarboxylase’s ability to bind the modified TPP?

Answer 2

Decrease drastically b/c boxed hydrogens would not be able to be removed, and would sterically clash with H on thiazolium ring

Question 3

Practice drawing sugars (questions on exam)

Answer 3

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Question 4

Pyruvate decarboxylase is able to bind this modified TPP normally, without any overall effect on the structure of the active site. What effect would the modified TPP have on the kcat of pyruvate decarboxylase?
Would it increase or decrease as a result of this modification?

Answer 4

Decrease drastically, would be unable to extract proton/form ylide, couldn’t start the reaction

Question 5

Pyruvate decarboxylase has one other essential cofactor besides TPP. Identify this second cofactor, and briefly explain its function

Answer 5

Mg2+ and it binds phosphates of TPP

Question 6

Know how to draw Haworth projections #3

Answer 6

-

Question 7

Non-reducing sugar

Answer 7

Hemiacetal and glycosidic bond so can NOT switch between beta and alpha

Question 8

Know structures of G6P

Answer 8

4a on exam

Question 9

Would G6P be able to return to original state? Has it undergone an irreversible step before water comes in to the mechanism?

Answer 9

Yes it would be able to
Refer to labelled being incorporated into G6P

Question 10

The ape Pongo stultividus contains two enzymes that are nearly identical in tertiary structure to
enolase, with some key differences. The first, Enzyme A, has replaced the two catalytic Mg? ions
with a single arginine. The second, Enzyme B, has replaced the two catalytic Mg2+ ions with a single
unprotonated glutamate.

Is it reasonable to suppose that Enzyme A catalyzes the same reaction as enolase? Explain effects this mutation would have on catalysis and substrate binding

Answer 10

Yes, Arg could polarize carboxyl of substrate as well

Question 11

Is it reasonable to suppose that Enzyme B catalyzes the same reaction as enolase? Explain effects this mutation would have on catalysis and substrate binding

Answer 11

No, b/c negative charge from glutamate would repel 2 phosphoglycerate
Binding would be almost impossible and if it did happen catalysis wouldn’t happen

Question 12

Alcohol dehydrogenase catalyzes conversion of acetaldehyde to ethanol. Enzyme has to add two hydrogens and two electrons to the carbonyl double bond of acetylaldehyde
One hydrogen is ultimately derived from the solvent. Where does the hydrogen come from?

Answer 12

NADH

Question 13

Alcohol dehydrogenase catalyzes conversion of acetaldehyde to ethanol. Enzyme has to add two hydrogens and two electrons to the carbonyl double bond of acetylaldehyde
Where do the two electrons come from?

Answer 13

NADH

Question 14

The residue of the following polysaccharides consist solely of unmodified glucose

Answer 14

Glycogen
Amylose
Cellulose
Amylopectin

Question 15

Suppose you incubate alcohol dehydrogenase with excess acetylaldehyde in the presence of D2O and stop the reaction after each enzyme has done exactly one reaction. You separate the enzyme, the ethanol, and the acetylaldehyde from each other (and the solvent), putting them each in separate fractions. Which fraction(s), if any, will contain D (labeled hydrogens)?

Answer 15

The ethanol fraction will contain D b/c oxyanion prior to formation of ethanol is protonated w/ an H+ from the external solvent via the proton relay system. H+ repalced w/D O-D
This isn’t completely right

Question 16

Treatment w/ EDTA affect catalysis of following enzymes?
Lysozyme

Answer 16

WOULD NOT b/c there are not metal ion cofactors involved in catalytic mechanism

Question 17

Treatment w/ EDTA affect catalysis of following enzymes?
Enolase

Answer 17

WOULD affect b/c would strip off 2 Mg2+ cofactors of enolase that polarized 2-phosphoglycerate to decrease pKa of proton that is to be extracted by K345

Question 18

Treatment w/ EDTA affect catalysis of following enzymes?
G6P

Answer 18

WOULD NOT affect catalysis b/c no metal ion cofactors involved in catalytic mechanism

Question 19

Treatment w/ EDTA affect catalysis of following enzymes?
Pyruvate Decarboxylase

Answer 19

WOULD NOT affect b/c no metal ion cofactors needed by enzyme for reaction mechanism. Portion of TPP is hidden within dimer interface, not exposed to solvent involved in reaction mechanism

Question 20

Treatment w/ EDTA affect catalysis of following enzymes?
Alcohol Dehydrogenase

Answer 20

WOULD affect b/c it would strip catalytic Zn2+ held by active site that polarized carbonyl of acetylaldehyde to make carbonyl carbon electrophilic

Question 21

Asp 52, there is single Cl- ion held in place a nearby Arg. This mutation does not impact the catalytic activity of the enzyme. Does existence of this mutant lend more support to the Phillips/SN1 model of lysozyme’s mechanism or to the Withers/SN2 model

Answer 21

Supports Phillips/SN1 model b/c in SN1 model Glu35 deprotonates H2O causing O of remaining OH- to attack and form bond with C1 of NAM of peptidoglycan forming beta anomeric carbon. Cl- is there to simply stabilize/balance the (+) charge of the oxocarbenium ion. In SN2, original Asp52 was directly involved in mechanism by attacking C1 of NAM to serve as leaving group. This mutants existence supports Sn1 b/c not affect catalytic activity of lysozyme so the Cl- must not be directly involvled in mechanism. DOES NOT support SN2 model b/c since it is likely not involved in catalytic mechanism, likely Cl- is not forming intermediate or ES complex with NAM.

Question 22

Binding cleft of P. stultividus lysozyme lacks any Trp, Phe, or Tyr, thought it is still able to cleave peptidoglycan at a normal rate

How would the lack of these amino acids affect the location of the scissile bond on peptidoglycan

Answer 22

Lacks Trp62 in site C of lysozyme binding cleft which normally sterically hinders NAM form binding to site C since NAM is too big to fit so that it can bind to site D so Glu35 and Asp52 can cleave scissile bond.
Without Trp62, NAM can bind to site C causing NAG to bind to site D instead of NAM causisng scissile bond to be after NAG rather than NAM

Question 23

Lack of these amino acids affect the formation of distorted half-chair substrate conformation

Answer 23

The half chair would be unaffected b/c those amino acids do not force the structure to form

Question 24

Lack of these amino acids affect the pH dependence of the enzyme’s activity?

Answer 24

pH dependence would be unchanged b/c Asp and Glu still would need to be deprotonated and protonated around pH 4-6