Exam #2 Material

Question 1

Know calculations of Vmax, Km, and Kcat

Answer 1

Vmax is 1/intersection @ y-axis
Km is 1/intersection @ x-axis
Kcat is Vmax= Kcat times {E}total

Question 2

Know calculations for alpha’ and Kmapp

Answer 2

Vmaxapp= 1/given concentration
Vmaxapp= Vmax/alpha’
Kmapp=Km/alpha’

Question 3

Inhibition effectiveness?

Answer 3

The smaller the Ki’, the more effective it is on enzyme activity. Math could be helpful in solving this question.
Reference question 1g

Question 4

Can you determine the kcats of both enzymes despite lacking sufficient amounts of enzyme?

Answer 4

Yes b/c saturating–> Etot–>Vmax–>Kcat

Question 5

The reaction velocity of Paukstelase is three times of Leease, which has a smaller Km?

Answer 5

You cannot determine this b/c Vmax DOES NOT correlate with Km

Question 6

Question 2c

Answer 6

What is Capp saying he aint pick an answer LJASDJAs

Question 7

Question 3a

Answer 7

Draw catalytic triad, do we actually need to know this question mark? Maybe have general idea?
Asp, His, and Serine

Question 8

Would any other serines on trypsin be covalently bonded to PMSF?

Answer 8

No b/c PMSF only interacts with activated/deprotonated serines

Question 9

Would PMSF be an effective inhibitor of elastase?

Answer 9

Yes, b/c PMSF acts on the catalytic triad which is identical

Question 10

Mixture of proteins. A= 32 kDa, B= 75 kDa, and C= 54 kDa. A is known to bind to the ligand Stukonthair, you decide to separate the proteins from each other using a column that has Stukonthair covalently attached to it. Identify the conditions you will use to elute protein A from the column, and explain the principle behind this elution. How do the identified conditions cause protein A to come off the column?

Answer 10

Elute w/ free Stukonthair, protein binds to free ligand instead of column
Change pH–> changes affinity for column

Question 11

Proteins B and C have no affinity for Stukonthair, so you are surprised to find that only protein B is in the Flowthrough of your affinity column. What does this face tell you about protein C?

Answer 11

C binds to A

Question 12

You repeat your purification (again by affinity chromatography), using a new mixture of proteins A, B, and C, but this time adding a Wash step that should allow you to get proteins A and C in different fractions. Identify the conditions used in this Wash step, and explain the principle behind them. How do the identified conditions cause proteins A and C to behave differently on the column?

Answer 12

Salt. Ions disrupt nonspecific interactions between A&C, so they separate

Question 13

Drawing gels on Lane

Answer 13

Refer to question 4d

Question 14

In which fraction would you expect to find protein A?

Answer 14

In Fraction 2 (61-90 kDa) b/c A and C interact so total would be 86 kDa

Question 15

Size exclusion chromatography be a reasonable way to isolate protein A from proteins B and C?

Answer 15

No b/c A and C would still be interacting and so they would be in the same fraction as B

Question 16

Rare ape Pongo stultividus, encounter a protease that, while similar in structure to chymotrypsin (same catalytic triad) has two novel features

The first of these novel features relates to the specificity pocket. At the bottom of the specificity pocket, where would you expect to find a Ser for chymotrypsin, you would find a Zn2+ (held there by two histidines). Based on this, identify two amino acids that you would reasonably expect this protease to cleave after

Answer 16

Asp and Glutamine (D and E)
Zn2+ ion–> positive charge –> negative things in the specificity pocket

Question 17

Second novel feature is that glycine whose backbone acts as a hydrogen bond donor in the oyxanion hole has been replaced by a proline. This substitution impacts neither of the overall protein folding, nor the presence of the oxyanion hole. What, if any, impact will it have on the rate of the reaction catalyzed by this protease?
Describe normal role played by oxyanion hole in thermodynamic terms (as related to the rate of catalysis), and how the mutation affects or doesn’t the ability of the oxyanion hole to perform the specified role?

Answer 17

Rate will be reduced
Oxyanion hold stabilizes transition w/ 2 H-bond donors
This would only have 1
T state would not be as stabilized
Kcat would be SMALLER

Question 18

Do expts on serine protease in deuterium labeled water (D2O). Once reaction is completed, where could you reasonably expect to find a deuterium instead of a normal hydrogen?
Yes or No
Ser
His
Asp
N-Terminal Amine
C-Terminal Carboxylic Acid

Answer 18

Ser: Yes
His: No
Asp: No
N-Terminal Amine: Yes/No
C-Terminal Carboxylic Acid: Yes

Question 19

“Adding a noncompetitive inhibitor to a reaction is kinetically the same as removing enzyme from it” What is your roommate talking about? Justify it in terms of how Km and Vmax are modified by adding the inhibitor and by reducing enzyme concentration

Answer 19

Non comp REDUCE Vmax, but does not CHANGE Km
Less enzyme would have the same effect

Question 20

Roommate now says “Wait, no, I meant an uncompetitive inhibitor- adding one of those is more like removing enzyme from a reaction” Why is he wrong in kinetic terms

Answer 20

Uncompetitive inhibitors also reduce Km–> less enzyme does NOT change this

Question 21

Best way to determine tertiary structure of enzyme Amorphase, enzyme is small only 10 kDa and contains abnormally large number of glycines. Friend says “Obviously NMR is the way to go. I doubt crystallography will work at all” Explain his reasoning with knowledge of amorphase primary structure

Answer 21

Glycine lacks sidechains so this amino acid can freely rotate around alpha carbon. Difficult to free/hold amorphase in one structure not moving b/c the many glycines within the primary structure will undergo rotation. So structure of amorphase will want to undergo conformational changes and cannot hold still for crystallography

Question 22

Consider oxyanion hole of substilisin, a serine protease
What interactions are provided by the oxyanion hold in serine protease catalysis?

Answer 22

Backbone nitrogen of serine protease active site form hydrogen bonds with oxyanion of tetrahedral intermediate of catalytic reaction

Question 23

Mutation of Asn to Ala in consideration w/ oxyanion hole. Greater impact on kcat or Km?

Answer 23

Kcat b/c the substrate already binded to serine protease active site, so this mutation affects the chemistry of the ES complex.
Target scissile peptide bond of protein to be destroyed

Question 24

Asn to Gln mutation. Effect on subtilisin’s activity?

Answer 24

Oxyanion hole stabilizes tetrahedral intermediate of serine protease catalysis by forming H-bonds using backbone nitrogens with oxyanion. Mutation will DECREASE subtilisin’s activity b/c Gln has longer sidechain, affecting the structure of the oxyanion hole and ability to form H-bonds. So no or little stabilizing of tetrahedral intermediate so enzyme cannot properly catalyze reaction to form products

Question 25

Mixture of proteins A, B, and C. A and B= 31 kDa and C= 92 kDa
Separate B from other proteins using size exclusion chromatography. Friend says awful idea won’t work. Explain friend’s reasoning.

Answer 25

Proteins A and B are same size so they are likely to elute together from the column. A and B are smaller than C, so they will elute last because they will get stuck inside pores of column resin bands

Question 26

You can isolate B from A and C using size exclusion chromatography. What did your friend now know (and you did) that made using this chromatography a good idea?

Answer 26

Some proteins behave as size of the larger quaternary protein complexes they are a part of with other proteins. Either A or B was “larger” than 31 kDa b/c they retained their larger quaternary complexes as such purification technique allows this

Question 27

A280 and Western Blot carried out perfectly w/ Protein B why?

Answer 27

Protein B must have little number of Tyr and Trp residues in its structure so did not absorb much 280 nm wavelength

Question 28

Anion Exchange Chromatography

Answer 28

Refer to Question #6 Fall 2022

Question 29

Eluted fraction Y in flowthrough, X in third eluted fraction, Z in fifth eluted fraction. X is 74 kDa, Y is 36 kDa, and Z is 130 kDa

Answer 29

Y has no (-) surface charges
X has no (-) surface charges (more than Y, less than Z)
Z has more (-) surface charges than X, so stronger attraction to DEAE

Question 30

All solutions were at pH of 9.5 and not the pH of 8.0 that was planned to be used. If all tertiary/quaternary structures remain intact, has using this buffer caused proteins X, Y, and Z to interact with/elute from the column differently than you expected in the previous question?

Answer 30

Will interact/elute differently b/c the pH 9.5 solution will cause DEAE to deprotonate causing the DEAE to become neutral. Proteins with (-) surface charges will not attract to DEA and in flow-through

Question 31

Protein Y inactivated by high salt concentrations. Will this be a problem given the above method of isolating protein Y?

Answer 31

No b/c protein Y has no (-) surface charges, so no attraction to (+) DEAE, so will end up in flow through anyways (elute first/fastest)

Question 32

Would TLCK irreversibly inhibit elastase and covalently link to elastase more often, less often, or as often as it is linked to trypsin?

Answer 32

No b/c lysine sidechain of TLCK would not go in the specificity pocket of elastase since elastase only cleaves peptide bonds after GLY, ALA, SER, and VAL (small hydrophobic amino acids). Lys is a hydrophilic and (+) charged amino acid

Question 33

TLCK

Answer 33

Draw catalytic triad
ASP HIS AND SER

Question 34

Would TLCK irreversibly inhibit chymotrypsin and covalently link to chymotrypsin more often, less often, or as often as it is linked to trypsin?

Answer 34

No b/c chymotrypsin cleaves only after PHE, TRP, TYR, and MET so lysine sidechain of TLCK will not fit in chymotrypsin specificity pocket

Question 35

You are studying an enzymatic reaction that follows Michaelis-Menten kinetics. Your experimental measurements of the Km:

Answer 35

Will decrease if you add an uncompetitive inhibitor to the reaction
Will increase if you add a competitive inhibitor to the reaction
Will stay constant when you decrease the amount of enzyme in the reaction
Will larger for substrates with a lower affinity for the enzyme

Question 36

The maximal velocity (Vmax) for a particular enzymatic reaction is:

Answer 36

Dependent on the concentration of the enzyme
Dependent on the kcat of the reaction
Independent of the Km of the enzyme for its substrates
Unaffected by a competitive inhibitor

Question 37

Consider the serine protease trypsin. It is true to say that:

Answer 37

The enzyme will cleave the peptide bond after a lysine
The amino acids in its catalytic triad are positioned in three-dimensional space in exactly the same way as the catalytic triad of elastase

Question 38

Measuring the concentration of protein by A280 depends upon the presence of this/these amino acid/s

Answer 38

Tryptophan
Tyrosine

Question 39

The first step of your purification for the enzyme answer selectase involved affinity chromatography. Afterwards you look at each fraction using the Bradford Assay. This method of looking at each fraction:

Answer 39

Gives you information about the quantity of total protein

Question 40

SDS-PAGE separates proteins:

Answer 40

Based on the mass of amino acids present
Using electric current