Lab chapter 6: Biochemical reactions, Enzymes 1

Question 1

Ribozymes are:

Answer 1

RNA molecules that act as enzymes

Question 2

Roughly one ounce of pure pepsin is capable of digesting/hydrolyzing two metric tons of ovalbumin in a matter of a few hours. This is an example of ____

Answer 2

an enzyme/catalyst speeding up a rxn which would otherwise be slow as fuck.

Question 3

The 3D shape of an active site is determined by:

Answer 3

the arrangement of the enzyme’s constituent amino acids

Question 4

Each amino acid consists of three groups, which are:

Answer 4

the central/alpha carbon is attached to the three groups: amino/NH2, carboxyl group, and side chain/R group

Question 5

What is the primary structure of a polypeptide?

Answer 5

the linear sequence of amino acids in a polypeptide chain

Question 6

Amino acids are linked together in a covalent bond referred to as a

Answer 6

peptide bond

Question 7

The secondary structure is made up of ____ bonds, which are formed between the ___ group of one amino acid and the ____ acids of another amino acid

Answer 7

hydrogen
hydrogen of amino + oxygen of carboxyl group

Question 8

The tertiary structure results from interaction of _____

Answer 8

side chains of its constituent amino acids and the surrounding solution
ex. water or a np substance

Question 9

A quaternary structure exists in proteins consisting of

Answer 9

more than one polypeptide chain

Question 10

The tertiary structure of a polypeptide or the quaternary structure of a protein are stabilized by three things, which are:
1.
2.
3.

Answer 10

1) hydrogen bonding between polar side chains

2) ionic bonds between positively charged and negatively charged polar side chains

3) covalent bonds between the sulfurs found in the side chains of two cysteine monomers (also known as disulfide bridges).

Question 11

Describe how a polypeptide will fold in water

Answer 11

will maximize interaction between nonpolar side groups, folding closer within the molecule

Question 12

The folding of a polypeptide in water results in the formation of

Answer 12

an active site having a specific size and shape. Note that the polar side chains are what makes up this active site because the nonpolar have folded in on themselves.

Question 13

Once the proper substrate has bound, an enzyme usually undergoes a conformational change known as

Answer 13

induced fit

Question 14

what occurs during induced fit?

Answer 14

puts stress on exisiting bonds within substrate that must be broken while fostering new bonds that must be made in order for a substrate to be converted into its product

Question 15

The amount of product in this experiment can be followed by
The amount of product formed can be quantified by

Answer 15

followed colorimetrically using a spectrophotometer
quantified by a standard curve

Question 16

describe what occurs in this experiment’s reaction,

Answer 16

The enzyme, acid phosphatase, binds p-nitrophenyl phosphate in its active site which allows a water molecule to be introduced between the phosphorus atom and the oxygen atom that links the phosphate group to the benzene ring, breaking (hydrolyzing) the covalent bond

Question 17

Describe the general procedure of the time course section

Answer 17

1. known amnt susbtrate placed into tube with acidic buffer solution
2. enzyme added at “time zero” then quickly mixed and a small sample (aliquot) is withdrawn
3. aliquot placed in alkaline solution, which stops reaction of that sample

Question 18

the longer the enzyme is allowed to work in the reaction vessel, the more product is formed over time, so long as

Answer 18

substrate concentration is not a limiting factor

Question 19

If aliquots of the reaction mixture are withdrawn at set time intervals and stopped as described above, one would expect

Answer 19

the color in each successive sample tube to be more yellow than previous

Question 20

The amount of product within each sample tube is directly proportional to

Answer 20

the amount of light absorbed by the tube’s contents.

Question 21

If one compares the amount of light absorbed by a sample tube to that of tubes containing known amounts of product, one can

Answer 21

quantify the amount of product contained within that particular tube. This comparison is simpli-fied by producing a graph known as a “Standard Curve.”

Question 22

Describe the important of the second time course

Answer 22

A second time course is run concurrently; this tube is identical to the first in all respects except that enzyme is not added. In place of the enzyme, an equal volume of deionized water is added to the tube. The second time course serves as a “control” (mentioned above) of enzymatic activity for any product in the control tube did not come about from the action of an enzyme!

Question 23

Describe general procedure of the “standard curve” exercise

Answer 23

a stock solution with a known product concentration is used to create a series of tubes in a process known as “serial dilution.” The contents of each of these tubes will absorb a different amount of a wavelength of light from the blue part of the visible spectrum.
Using a spectrophotometer, one can obtain a number which corresponds to the percentage of the initial light that is absorbed by a solution as it passes through.
Once absorbencies are obtained for a series of known concentrations of product, a graph of those results can be plotted. This allows the user to obtain a product concentration for any absorbance value that falls within the range of that graph

Question 24

you obtain an absorbance value for a given tube in the time course, you

Answer 24

can determine how much substrate was converted to product within that time period by using your standard curve.

Question 25

the microcentrifuge tubes in the time course procedure are ___mL

Answer 25

1.5 ml

Question 26

Add ____ uL of 0.1M NaOH to each of the experimental and control sample tubes

Answer 26

200

Question 27

____uL of citrate buffer (pH of 5.0) is added to the E-rxn and C-rxn, and ___ uL of 0.006 M p-nitrophenyl phosphate (substrate)

Answer 27

400uL for both

Question 28

In the enzyme reaction tube,

___uL of buffer, ___uL substrate, and ___uL of enzyme is added

Answer 28

400uL of each

Question 29

In the C rxn tube, ___uL of buffer, ___uL substrate, ____ uL deionized H2O

Answer 29

400

Question 30

In the E/C-0/10/20/30/60, ___uL NaOH and __uL rxn mix is added at the allotted time and mized

Answer 30

200uL NaOh and 200uL of rxn mix

Question 31

___uL of each sample is added to the 96-well plate in the spectrophotometer

Answer 31

300

Question 32

Give a general summary of serial dilution, steps 1 through 6

Answer 32

100uL of deionized H2O to 2-5
100uL of 0400 p-nitrophenol to tubes 1 and 2, creating a 1:2 dilution

Close cap and mix contents of p200. Withdraw 100uL of p200 and add it to tube 3

Close caps and mix contents of p100. withdraw 100uL and add to tube 4

etc etc.
discard in biohazard

Question 33

After the p200/100/50/25 is added, add __uL citrate buffer, ___uL H2O, and ___ uL NaOh

Answer 33

100uL, 100uL, 300uL

Question 34

After the citrate buffer, H2O, and NaOh is added to the tubes, add ____, ___, and ____

Answer 34

100uL citrate buffer (pH 5.0)
100uL deionized water
300uL NaOH

Question 35

The caps must be inverted ___ times to mix the contents

Answer 35

three

Question 36

After mixing is completed, ___uL of each product should be transfered to 96-well plate

Answer 36

300

Question 37

A standard curve is a graph in which

Answer 37

known concentrations of solute are plotted against their respective measured optical absorbancies, obtained using a spectrophotometer. In theory, a standard curve should be linear.

Question 38

the x-axis of the graph is used for ____ and the y-axis is used for

Answer 38

solute concentration
optical absorbance (density)