Biochemistry I Test 2 Review Answers
Name the 6 E.C. Classes of enzymes. List the reaction mechanisms.
3.Ligase A + B + ATPßàAB + ADP + Pi (Synthesizing something using ATP, 2:1)
4.Lyase A(X)B(Y) ßàA=B + X-Y (Forward reaction breaking down 1:2; backward reaction synthesizing 2:1)
5.Isomerase A(X)B(Y) ßàA(Y)B(X) (rearranging on the SAME compound) - Can be potentially a MM enzyme
You can identify a lyase catalyzed reaction by the addition to or formation of a ______________. A double bond. Also, it goes from 1 -> 2 parts in the forward reaction (breaking down). Or 2 parts -> 1 part in the backward reaction (synthesis).
Does a hydrolase break things down or put things together? Break things down with the addition of water
-O2CCH=CHCO2- + H2O -> -O2CCH(OH)CH2CO2- Lyase
Phosphofructokinase (Or any kinase) Transferase (Transferring phosphate group from something like ATP)
Fructose-6-PO4 + ATP -> fructose-1,6-bisPO4 + ADP Transferase (Pi from ATP is transferred and becomes part of product, not released)
(OH group moved around)
Which one of the following enzymes is categorized as a hydrolase?
What are enzymatic RNA molecules called?
What is an example of one that acts as a transferase and also exists on ribosomes to help form peptide bonds? Peptidyl Transferase Enzyme Kinetics.
What are the two types of enzyme kinetics discussed? What do they assume about the enzyme/substrate interaction?
What is the rate constant for the association of ES from E + S? k1
What is the rate constant for the dissociation of ES to E + S? k-1
What is the relationship between these two rate constants? (Directly proportional versus inversely proportional) Inversely proportional
What is the rate constant for the association of E + P from the ES complex? K2
As mentioned before, in RAPID EQUILIBRIUM, k-1 and K1 >>>>>> k2. (K-1 and k1 are much greater than k2). If you assume this, what is the smallest value rate constant? K2
What is the slowest step then? (E + S to ES, or ES to E + P) ES to E+P
Which of the rate constants is considered the rate limiting constant? K2
The Rate limiting constant = catalytic rate constant = turnover number = ____k2____________
What is k1/k-1 also called? ____Ka______ (dissociation/association) rate constant
What is k-1/k1 also called? _______Ks___ (dissociation/association) rate constant
How do you find Vmax? (As a function of rate limiting steps and enzyme concentration)
Vmax = [E] total x K2 (or Kcat same thing)
An 5x increase in [E]total would have what effect on Vmax? X5
How do you determine enzyme efficiency? Kcat/Km or K2/Km
What is the maximum enzyme efficiency? (Considered a “perfect” enzyme) Why can’t it go any higher/What is it limited by? 10^9, limited by the maximum rate of diffusion of substrate to enzyme.
3 Michaelis Menten Equations (Assumptions)
Combining these 3 equations give you the overall MM equation. What is this? (Uses Vo, Vmax, Km or Ks, [S]) When can Km be used interchangeably for Ks?
Vo/Vmax = [S]/ (Km + [S])
Km = Ks when K2 is very small. You can see this because Km = (k-1 + k2)/k1. When k2 is very small, I set it equal to 0. Then Km = k-1/k1 = Ks, your dissociation constant.
Briggs Haldane (Steady state)
What is Km? What does it show about the ability of the enzyme to binding to substrate? How do you define it in terms of [S]? In terms of rate constants?
What is the cellular [S] of any organic compound usually going to be? Km. Because Km represents [S] at half Vmax, it is good for a intracellular compound to exist at Km because it has room to modulate substrate concentration.
This is as opposed to having [S] when Vo is at Vmax.
Line-Weaver Burk Plot = Double Reciprocal Plot.
What is the equation for a Line-Weaver Burke Plot? (In terms of [S], Vo, Km) 1/Vo = (1/[S]) (km/Vmax) (1/[S]) + 1/Vmax
What is the shape of a Michaelis Menten enzyme plot on a kinetic graph of [S] vs. Vo? What is the shape of an allosteric enzyme?
Michaelis Menten is the hyperbolic. The allosteric enzyme is the sigmoidal (S) curve.
You need to know the equation for Km apparent for all types of reversible inhibitors. For competitive inhibitor, Km increases (because you need more substrate to reach ½ Vmax, so Km apparent = Km true (1 + [I]/Ki).
Km app = (10^-6) ( 1 + (10^-5/10^-3) -> Km app = (10^-6) (1+100) = ~10^04
Vmax stays the same for competitive inhibition.
In Classic noncompetitive (Alpha = 1, Ki values for EI to I versus ESI to ES are the same), Km stays the same.
25 uM. You can use MM equation, or you can see that Vo is ½ Vmax in this scenario.
Vmax = 210 umol/min
Use MM. Vo/Vmax = [S]/(km + [S]) Plug and Chug.
70/Vmax = .5km/ (km + /5km)
70/Vmax = .5km/1.5km = 1/3 -> Vmax = 210 umol/min
Use MM. Vo/Vmax = [S]/(km + [S])
.1Vmax/Vmax = [S]/(Km + [S]) = 1/10 = [S]/(km + [S])
Rearrange -> km + [S] = 10[S] -> km = 9[S] -> [S]/km = 1/9
Efficiency = kcat/km = 10^3/(2 x 10^-3) = .5 x 10^6 = 5 x 10^5 Max efficiency is 10^9.
Max efficiency/efficiency = 10^9/(5 x 10^5) = .2 x 10^4 = 2 x 10^3 times less efficient
See chart for types of reversible inhibitors and properties (Noncompetitive (classic/mixed), Uncompetitive, Competitive)
Listed are SPECIFIC reversible inhibitors. What type of inhibitors are these (comp, noncomp, uncomp.)?
Explain what compound they mimic, what reaction they stop from taking place, what enzyme they inhibit, and what possible product forms from the inhibition.
Anti-free is toxic because alcohol dehydrogenase participates in the conversion of ethylene glycol in the anti-freeze to oxalic acid, which precipitates in the kidneys. The same enzyme is responsible for the toxicity of methanol by converting methanol to….
Sulfa drugs are analogs of PABA. They function by…
Viagra works by…
In answer choice E, what type of enzyme classification catalyzes the hydrolysis of cGMP?
What type of a bond does an irreversible inhibit make with the active site of the enzyme? Covalent
What are some types of nerve gases and organophosphorus insecticides that act as irreversible inhibitors?
Sarin (nerve gas), DFP (nerve gas), Parathion (insecticide), Malathion (insecticide). Note: We are not affected by insecticides because we metabolize the compound differently. Insects oxidize it with microsomal oxidase whereas humans hydrolyze it. Insecticides are an organophosphorous compound (Big P in the middle) with an S bound to it. In insects, the S is replaced by an O.
They work by irreversibly inhibiting the breakdown of the ___acetylcholine (Ach)_______ neurotransmitter by ___acetylcholinesterase_______ (enzyme).
The enzyme listed above has a ___serine__ (A.A.) residue in its active site. Phosphorylated this will block the active site.
What is an example if a reversible (Reversible not irreversible, sorry)inhibitor that is also known to be a male enhancement drug? Viagra
What enzyme and substrate reaction does it inhibit? What happens to the substrate during the reaction? What is the role of the original substrate, physiologically? Substrate cGMP is supposed to react with enzyme phosphodiesterase, is broken down. Role of cGMP is relaxation of smooth muscle in walls of blood vessels, improving erection. Viagra prevents the breakdown of cGMP by binding to phosphodiesterase instead.
What is an example of an irreversible inhibitor that targets bacteria cell walls?
Draw and describe the structure of peptidoglycan, the compound which forms bacterial cell wall.
Describe the reaction used to form a bond between the pentaglycine bridge and the 4 residues. What enzyme catalyzes this reaction? Does the enzyme attack the D-alanine itself or the peptide bond between one D-Ala and another? Glycoprotein Peptidase, it attacks the PEPTIDE BOND
Penicillin is an irreversible inhibitor of the enzyme ___glycoprotein peptidase______ which catalyzes a step in bacterial wall synthesis.
Penicillin is also considered a “suicide substrate”. Why?
Because when the enzyme binds to the substrate (inhibitor) through a covalent bond, it is irreversibly inhibited. Almost as if the enzyme is committing “suicide”.
The structure of penicillin contains a __Beta-lactam______ ring, which has a reactive __N-C (peptide)___ bond that mimics the same bond between D-ala – D-ala.
?: The organophosphorus nerve gases, such as sarin, and insecticides, such as malathion,
What’s the difference between bisubstrate reactions and single substrate reactions (this is not a trick question)?
Bisubstrate – 2 substrates forms 2 different products. Used in class was substrate A (forms Product P) and substrate B (forms product Q)
Single substrate – only 1 substrate and one product. We only discussed single substrate reactions in terms of the enzyme kinetics due to addition of reversible inhibitors (noncomp, uncomp, competitive)
Bisubstrate reactions can differ in the enzyme mechanism. What are the two types of displacement that can occur during a bisubstrate reaction? Please list mechanism steps.
(not E). It binds, the enzyme goes back to normal, product Q is released. E’ (original) -> E (normal). There is a “double” displacement because product is being displaced at 2 separate times, first P is leaving. Later Q is leaving.
Which one occurs when both substrates bind to the enzyme before products are formed?
Single displacement either random or ordered
Which one occurs where two substrates bind and react separately? What is another name for this bisubstrate reaction?
Double Displacement. Ping Pong Bi Bi
Please draw the Line-Weaver Burke Plot for each type of bisubstrate reaction. Remember the X axis label is different than a plot for reversible inhibitors. Also draw kinetic lines in order of increasing [B] (substrate B concentration). (LEFT is Double displacement, RIGHT is single displacement) Notice the changes in Km and Vmax…
The Line-Weaver Burke plot for the Ping Pong BiBi reaction looks very similar to a LWB plot of a
__uncompetitive_____ inhibitor in a single substrate reaction. (Both a set of parallel lines)
The Line-weaver plot can distinguish if a bisubstrate mechanism is a ___single______ versus ___double_________ displacement. The line-weaver plot can also distinguish if a single substrate enzyme mechanism has been affected by __noncompetitive (mixed or pure)____, __competitive___, or __uncompetitive____ type of reversible inhibitors.
What type of bisubstrate reaction is this and why?
Single random displacement. Because either A or B can bind first
So… what if you are a scientist and you observe a reaction, but you do not know if it is a single or double displacement type mechanism. What 3 techniques can you use to figure this out?
2.___Substrate Binding Assay____ – what do you use to distinguish if substrate addition is randomed or ordered?
(How do you determine # of binding sites and Ks?) You can use Scatchard Plot
Btwn single and double displacement
A Scatchard Plot shows __[S]bound______ in the X-axis and _[S]bound/[S]free____ in the y Axis.
What is it used to determine? (2 things)
# of binding sites and Ks
Chapter 14 Enzyme Mechanisms
Enzymes work to catalyze the reaction. Speeding the rate of reaction.
Is ΔG of the reaction affected? No
Is Keq affected? No
Is Arrhenius activation energy affected? Yes, it is lowered. Look at the difference of free energy from your transition state (X) and your enzyme catalyzed transition state (EX). That’s the amount it was lowered by.
List the 7 reasons for catalysis by enzymes
It’s just one way that it can be destabilized)
Why do pharmaceutical companies most often use transition-state analogs are enzyme inhibitors? Rather than a substrate? Because TSA will compete with the substrate and bind better.
EX: CHYMOTRPYSIN (an enzymatic cleavage after the following amino acids: __Phe___, ___Trp___, _Tyr____ and
____Leu___) has a __Ser_____ (A.A.) in its active site. The geometric conformation of the susbstrate is at first ______trigonal planar______. After the serine attacks the substrate (usually at the carbon of a carbonyl group), it forms an _______oxyanion_________ intermediate, which has ______tetrehedral______ geometric structure (think what is sp3 hybridized).
Near Attack Conformations (NACs)
They are within __3.2 Angstroms = .32nm___ (length – give units) and approach angle of __+/- 15___ (degrees) of the bonding angle of the conformation of the transition state.
The dotted line is if the ES is never destabilized by anything and falls into the energy well, making it hard to overcome it.
What are the reactive standard amino acids? (8)
Cys (-SH), Ser (-OH) Tyr (-OH) Thr (-OH) Glu (COOH) Asp (COOH) Lys (NH3+) His (C—NH+---C) - What reactive standard amino acid is involved in each of these enzymes?
Which of these, when reacted with an aldehyde, forms a Schiff base? Aldolase (it has a lysine in it’s AA)
Which of these in present in papaya? Papain
3 reactive amino acids can be used to form a Charge Relay System, also know as a _____Catalytic________ Triad. For example, in SERINE Proteases (a protease is an enzyme that cleaves proteins. Serine signifies that serine is the reactive amino acid in the active site of the protease), what is the catalytic triad consist of? (Please Draw too)
Which amino acid forms the tetrahedral intermediate with the substrate/peptide? Serine
Which amino acid acts as a general base to remove a H+ from the OH groups of one of the other amino acids?
Which amino acid acts to stabilize orientation and structure of triad by H bonds and charge-charge interaction?
What amino acids can act as a general acid catalysis? (which amino acids are acidic or have a proton to donate?)
Aspartic Acid, Glutamic Acid, Histidine, Serine, Tyrosine, Threonine
What amino acids can act in general base catalysis? His, conjugate base of Asp, Glu,
On the left is a mechanism of an aspartic protease. Notice how many aspartate residues there are (one on each side). 2 residues
What type of bonds do the arrows point at? Are these permanent or transient? Would you could them having a high barrier or low barrier to form/uniform? H bonds, Transient, low barrier
Notice the two circled items. Under FT, The N of the substrate is taking an H away from aspartic acid. In this aspartic acid acting as a base or acid? Under EP’Q, is the aspartate residue acting as a base or acid?Acid, base
Aspartic Proteases employ _Low Barrier Hydrogen Bond_
(type of bonds) in their mechanism.
They also use the 2 Asp residues in general base/acid_ catalysis.
HIV protease is needed to cleave the polyproteins formed after translation of mRNA from HIV genome. The results of this cleavage is later integrated into the host cell chromosome. So basically important for HIV viral replication.
geometry of the TS.
To be used as a catalysis, Zinc is anchored by 3 bonds to what type of amino acid residue? 3 Histidines
Zinc reacts with water to form a 4th bond, attached to a __OH_____ (certain functional group…. The same present on serine and other reactive amino acids)
Metal ion catalysis can…
Ch 4-5. Protein Sequence Analysis
You can cleave a protein into oligopeptides in 2 ways: enzymatic cleavage OR chemical cleavage.
Please write the name of the cleaving agent and after or before what amino acids it may cleave at.
3.BNPS-Skatole; after Trp
****NOTE: NO ENZYMATIC CLEAVAGE IS POSSIBLE BEFORE OR AFTER PROLINE*****
To remove an amino acid from a protein one step at a time… you can use Edman degradation. From which terminus is the amino acid removed from?
Chapter 6 Protein Structure.
Name your 4 levels of protein structure and what composes each.
What are post-translational modifications to amino acids considered under? Primary
What are disulfide bridges considered under? Primary
Under what circumstance will the 3rd level of a protein structure exist also as the 4th level? If the protein is comprised of only one subunit. Like myoglobin.
What is the bond between the C carbonyl-N shown here called?
This bond has a partial ____double______ bond character. Does this allow for any rotation? No
What geometric configuration would you label the groups of the peptide bond? (planar, tetrahedral, etc) Planar
Can the N of the peptide bond engage in acid/base reactions? (can it donate or accept a proton)? No
Can it form H bonds? Yes
The peptide bond and atoms around it will have a ___trans_______ (cis/trans) configuration.
On a polypeptide chain, about what bonds can rotation can occur? What are the angles represented by the N-C alpha bond and the C alpha – C carbonyl bond called?
Phi – N-C Alpha
Psi – C alpha – C carbonyl (Psi as a Greek letter looks like a trident)
A Ramachandran diagram shows sterically reasonable values of the phi (X axis) and psi angles (y axis). In quadrant 2, the phi angle is <0, and the psi angle is >0. What type of secondary structure predominates?
Either parallel or anti-parallel Beta sheets
In quadrant 3, the psi angle is <0 and the phi angle is <0. What type of secondary structure predominates? R-handed alpha helix
Why is the amino acid glycine not counted in these angles?
Glycine would show up everywhere, thus negating the purpose of the plot in the first place, to see the predominance of certain values of phi and psi angles.
Glycine shows up everywhere because 1) it’s side chain is really small. 2) it is not chiral. Because of these two reasons, glycine is not likely to cause any steric hindrance in any which way rotation of its phi or psi angle -> allowing for a large range for sterically-okay phi and psi angles.
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