Biochemistry Midterm 1

Front 1

Which are affected by the catalytic activity of enzymes?

Back 1

a) Transition state
b) Rate constant for the reaction
c) Standard free energy of activation
d) Rate at which the reaction reaches equilibrium

Front 2

Describe catalytic mechanism triose phosphate isomerase...

Back 2

a) Acid-base catalysis: H+ ions are transferred between enzyme and substrate
b) H+ is pushed in addition to electron pairs
c) Not a redox reaction – No covalent bonds are formed
d) No hydrolysis occurs

Front 3

Catalytic mechanism for Chymotrypsin?

Back 3

a) Protease – catalyzes hydrolytic cleavage of peptide bonds
b) Uses covalent catalysis, general acid-base catalysis, and transition-state stabilization

Front 4

Two compounds pyruvate could be converted into under anaerobic conditions (excluding cellular resp.)

Back 4

a) Lactate fermentation
i) Lactate dehydrogenase – enzyme
ii) NADH + H  NAD+
b) Alcohol fermentation
i) Acetaldehyde then ethanol
ii) Pyruvate decarboxylase (releasees CO2) and irreversible
iii) Alcohol dehydrogenase – NADH + H  NAD+

Front 5

How many substrate-level phosphorylation steps does glycolysis have?

Back 5

2

Front 6

Substrate-level phosphorylation...

Back 6

Formation of ATP by phosphoryl group transfer from a substrate.

Front 7

How many C in glucose?

Back 7

6

Front 8

How many C in pyruvate?

Back 8

6

Front 9

How many pyruvate molecules produced by a single glucose?

Back 9

2

Front 10

“Gateway” step of glycolysis?...the "Ike" of Glycolysis?

Back 10

a) Step 3
b) Fructose 6-phosphate<->Fructose 1,6-bisphosphate
c) Phosphofructokinase is the gateway!!!!!

Front 11

Describe Phosphofructose kinase-1 mechanism...

Back 11

*highly allosteric regulated enzyme - activity increased when cell’s ATP supply is depleted or when ATP breakdown products, ADP and AMP accumulate – enzyme is inhibited whenever the cell has ample ATP
*an allosteric enzyme made of 4 subunits and controlled by many activators and inhibitors. *catalyzes the important "committed" step of glycolysis, the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP.

Front 12

Linewaver-Burke plot

Back 12

*Reciprocal of Michaelis-Menten used to plot Lineweaver-Burk plot
i) X-axis – 1/[S]
ii) Y-axis
1. Competitive 1/V0 = Vmax[S]/alphaKm + [S]
2. Uncompetitive 1/V0 = (Km/Vmax) 1/[S] + alp’/Vmax
3. Mixed inhibition 1/V0 = (alpKm/Vmax) 1/[S] + alp’/Vmax

Front 13

Competitive inhibitor

Back 13

Competes with substrate for the actie site of an enzyme, forms EI complex which does not lead to catalysis, because it is reversible – competition can be biased...
i) High [S] – normal Vmax
ii) [S] which V0 = ½ Vmax increases in the presence of inhibitor by factor of alpha

Front 14

Uncompetitive inhibitor

Back 14

Binds at a site distinct from the substrate active site and binds only to ES complex
i) High [S], V0 approaches Vmax/alp’[S] – lowers measured Vmax and apparent Km

Front 15

Enzyme inhibitors can bind...?

Back 15

Reversibly or Irreversibly

Front 16

Allosteric enzymes function through...

Back 16

REVERSIBLE noncovalent bonding of regulatory compounds called allosteric modulators/effectors

Front 17

Enzyme inhibitors always act to reduce the velocity of the reaction catalyzed by the enzyme but in different ways...

Back 17

either competing with the enzyme to form EI complex or changing the shape of the substrate

Front 18

Allosteric effects DON’T always...

Back 18

reduce velocity of reaction, homotropic effectors act to increase the rate by creating a better fit after conformational change

Front 19

Heterotrophic allosteric effectors bind to a separate...

Back 19

“regulatory” subunit which may have a positive or negative effect on the enzyme – so not all allosteric effectors bind on the active site

Front 20

Allosteric enzyme plots...

Back 20

*produce a sigmoid saturation curve rather than hyperbolic curve of typical non regulatory enzymes
*K0.5 and [S]0.5 used instead of Km

Front 21

Homotropic allosteric enzymes generally are...

Back 21

Homotropic allosteric enzymes generally are multi- subunit proteins and the same binding site on each subunit functions as both the active site and the regulatory site.

Front 22

In Homotropic allosteric enzymes the substrate most commonly acts as...

Back 22

*Most commonly, the substrate acts as a positive modulator (an activator), because the subunits act cooperatively: the binding of one molecule of substrate to one binding site alters the enzyme’s conformation and enhances the binding of subsequent substrate molecules.
*This accounts for the sigmoid rather than hyperbolic change in V0 with increasing [S]. In Sigmoid kinetics, a small change in [M] can be associated with large changes in activity

Front 23

Heterotropic Allosteric enzymes...

Back 23

Different responses to their substrate-activity curve because some have negative inhibitor modulators (more sigmoid substrate-saturaion curve with increase in K0.5) (b, -), positive activating modulators (may cause curve to be more hyperbolic with decrease in K0.5 but no change to Vmax) (b, +), and some may have both! --- Graphs: (b) central curve shows the substrate-activity relationship without modulator. (c) A less common modulation, in which Vmax is altered and K0.5 is nearly constant

Front 24

Noncompetitive Inhibitors bind to...

Back 24

a siter outside the active site, destroys some enzymes so it decreases Vmax but does not change Km

Front 25

Feedback inhibition is an example of...

Back 25

enzyme regulation by an allosteric effector that turns off enzyme activity

Front 26

Michaelis-Menton equation...3 assumptions

Back 26

1. V0 – no product has been formed yet so there’s no k-2¬ reaction
2. The conversion of ES to E and product is one step
a) k¬cat is rate-determining slowest step
3. Steady State assumption
a) Rate forward (k1) equals the rate backward (k-1)

Front 27

The lower the value of Km...

Back 27

The lower the value of Km, the more efficient the enzyme is with that substrate

Front 28

The larger the k1, the smaller the k-1, the...

Back 28

The larger the k1, the smaller the k-1, the better the substrate and enzyme come together

Front 29

The higher the Vmax the...

Back 29

The higher the Vmax the better the substate

Front 30

Lower kd means...

Back 30

Lower kd means higher binding affinity
(k1/k-1)

Front 31

Oxidation state for C atom

Back 31

a) Carbon loses more electrons to its neighbors – oxidation increases
b) More Oxygen, more oxidation

Front 32

Reaction rate

Back 32

V = k1 [A1] [A2]

Front 33

Chymotrypsin mechanism Step 1

Back 33

*Substrate binds to chymotrypsin with side chain of residue adjacent to peptide bond to be cleaved in a hydrophobic pocket

Front 34

Chymotrypsin mechanism Step 2

Back 34

*Ser195 attacks the peptide carbonyl group, breaking the double bond and forming a short-lived negative charge on carbonyl by pulling H+ off Ser

Front 35

Chymotrypsin mechanism Step 3

Back 35

*Instability of negative chg leads to collapse of tetrahedral intermediate; reforming the double bond with C, displacing C-NH bond – breaks peptide bond and AMINO group leaves and is protonated by HIS57

Front 36

Chymotrypsin mechanism Step 4

Back 36

*incoming water molecule is deprotonated (H+ gets pulled off_ by general-acid catalysis, attacks hydroxide on the ester linkage of the acyl-enzyme which generates a second tetrahedral intermediate with oxygen in the oxyanion hole w/ neg. charge

Front 37

Chymotrypsin mechanism Step 5

Back 37

*collapse of tetrahedral intermediat forms second product – a carboxylate anion and displaces Ser195
*Diffusion of second product from active site regenerates free enzyme

Front 38

Chymotrypsin Mech Step 1-5 Summarized

Back 38

Substrate binds -> Induced fit -> Asp 102 – His 57 Hydrogen bond is compressed -> His 57 deprotonates Ser 195, making it a better nucleophile -> Ser 195 attacks carbonyl -> tetrahedral intermediate -> acyl enzyme intermediate -> His 57 depronates water -> water attacks -> tetrahedral intermediate -> products.

Front 39

What if serine was substituted with the nonpolar alanine?

Back 39

Because alanine is nonpolar a site-directed mutagenesis on chymotrypsin so that serine is ssubstituted with alanine would affect the 2nd step, alanine would not be able to attack the peptide carbonyl bond. H+ would not be pulled of alanine, and this would halt the rest of chymotrypsin mechanism

Front 40

Steady state assumptions equation

Back 40

a) k1([Et] – [ES]) x [S] = k-1[ES] + k2[ES]
b) in terms of reaction velocities

Front 41

Glycolysis coversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, and then to 3-phosphoglycerate...

Back 41

insert pic or write out mechanism / reaction description...

Front 42

Types of Reactions

Back 42

a) Oxidoreductase – transfer of electrons (redox), includes dehydrogenase
b) Transferases – move chemical groups between two different substrates (includes kinases), always 2nd order reactions with atleast two substrates
c) Hydrolases – hydrolysis “cut with water” (sever covalent bonds, etc)
d) Lyases – form or remove carbon-carbon bonds by addition/removal of chemical group
e) Isomerases – move chemical groups inside one substrate
f) Ligases – form single bonds between a variety of different atoms; C-C, C-O, C-S, C-N (condensation reactions) – ATP provides energy for bond formation (DNA ligase is an example)

Front 43

Classes of amino acids

Back 43

*Nonpolar aliphatic R groups (7)
Alanine, Guanine, Isoleucine, Leucine, Methione, Proline, Valine

*Aromatic nonpolar R groups (3)
Phenylalanine, Tyrosine,Tryptophan

*Polar Uncharged R groups (5)
Aspagine, Cysteine,Glutamine, Serine, Threonine

*Positively charged Basic R groups(3)
Histidine, Lysine, Arginine

*Negatively charged Acidic R groups (2) Asparatate & Glutamate