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Biochemistry 1 Test 2

Biochemistry I Test 2 Review Answers

Name the 6 E.C. Classes of enzymes. List the reaction mechanisms.

  1. Oxidoreductase A- + B ó A + B- (redox reactions, when electrons move around)
  2. Transferase A-B + C ßà A + B-C (transferring a group from one compound to another 2:2)

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 

  1. Hydrolase A-B + H2O ßàB-OH + A-H (breaking down (hydrolysis) with water)

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). 

  • A synthase and a synthetase both synthesize compounds, the difference being a synthetase uses a high energy phosphate compound where as the synthase does not.
  • Typically the lyase-catalyzed reaction happens in reverse. It synthesizes a new product because it makes 1 piece from 2 pieces. However, it does not use ATP to do this. Because of this you can also consider a lyase as a __________. Synthase
  • Ligase synthesizes a product from 2 reactants using ATP as an energy source. Because of this you can also consider ligase as a _______. Synthetase

Does a hydrolase break things down or put things together? Break things down with the addition of water

  • You can identify a hydrolase-catalyzed reaction by the addition of a __________ molecule and also because the reactant is ___________ (1 part -> 2 parts). H20, Being broken down
  • How many reactants does an isomerase involve? How many products form? 1:1
  • The presence of water in a reaction does not necessarily mean it is a hydrolase catalyzed. When can you tell it is a hydrolase? When can you tell it is a lyase? For hydrolase - a cleavage will occur with the addition of For lyase - Something can be cleaved along with the formation of a double bond on a product. BUT water can be added to the double bond in the reverse reaction with the synthesis of a productIdentify the enzyme that catalyzes the following reactions:

-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)

Isomerase

(OH group moved around) 

Which one of the following enzymes is categorized as a hydrolase?

  1. Trypsin
  2. Glucokinase
  3. V8 Protease
  4. A + C
  5. All of the above
  • and C are proteases, enzymes that catalyze breakdown of peptide bonds through hydrolysis.
  • is a kinase -> transferase.

What are enzymatic RNA molecules called?

Ribozymes

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?  

  1. Briggs Haldane – Steady State – Rate of ES formation equals rate of ES breakdown. [ES] stays constant
  2. Michaelis Menten – Rapid Equilibrium – k1 and k-1 >>>>> k2. Substrate binding step occurs very quickly. Catalytic step not so much.

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)

  1. Vo = k2 [ES]
  2. [E] total = [E] free + [ES] bound
  3. Keq = ([E]free [S])/ [ES] = k-1/k1 = ks

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)

  • Rate of ES formation = rate of its _________breakdown_________
  • So [ES] stays __constant_______ (constant or dynamic?)

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? 

  • Km refers to binding affinity. Low Km is higher binding affinity. Because it can be equated to Ks, a lower Ks (a lower Km) would lead to a higher Ka (association) which would have a better affinity - Km is the [S] at which Vo is equal to ½ Vmax.
  • Km = (k-1 + k2)/k1

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. 

?’s.  

  1. Which listed below does not correspond to a major class of enzymes outlined by International Union of Biochemistry?
  2. Hydrolases
  3. Transferases
  4. Carboxylases
  5. Isomerases

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

  1. Competitive inhibitor (Ki = 1 x10^-5 M) binds to an enzyme that has a Km = 1 x 10^-6 M for its substrate and a Vmax of 1 x 10^-4 moles/min. Calculate Km apparent value with 1 x 10^-3 M inhibitor. 1 x 10^-7 M B. 1 x 10^-6 M C. 1 x 10^-5 M D. 1 x 10^-4 M
    1. 1 x 10^-3 M
  1. What is the Vmax apparent of the enzyme with the competitive inhibitor in #2?

Vmax stays the same for competitive inhibition. 

  1. 1 x 10^-7 mol/min B. 1 x 10^-6 mol/min C. 1 x 10^-5 mol/min D. 1 x 10^-4 mol/min
    1. 1 x 10^-3 mol/min

In Classic noncompetitive (Alpha = 1, Ki values for EI to I versus ESI to ES are the same), Km stays the same. 

  1. If the inhibitor in #2 is a classic noncompetitive inhibitor, what is the Km apparent? 1 x 10^-7 M B. 1 x 10^-6 M C. 1 x 10^-5 M D. 1 x 10^-4 M
    1. 1 x 10^-3 M
  1. If Vmax = 120 umol/min, and Vo = 60 umol/min at 25 uM substrate [S] for an MM enzyme, what is its Km?

25 uM. You can use MM equation, or you can see that Vo is ½ Vmax in this scenario. 

  1. For an MM enzyme, what is the Vmax if vo = 70 umol/min when [S] = .5Km?

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

  1. What is the ratio of [S]/Km when velocity of an enzyme catalyzed reaction is 10% of Vmax?

Answer: 1/9

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

  1. If the turnover number is 1 x 10^3 S^-1 and the Km for an enzyme is 2 x 10^-3, how much less efficient would the enzyme be than the best known enzymes/perfected enzymes? (You need to know the max efficiency value of a perfect enzyme. You also need to know what rate constant stands for turnover number)

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

  1. Which of the following statements is true about Michaelis-Menten enzymes?
    1. They never have more than one subunit
    2. They always follow rapid equilibrium kinetics
    3. They never have allosteric effectors
    4. A and C
    5. All of the above
  2. The Briggs Haldane steady state assumption rests on the premise that The concentration of ES complex does not change
    1. Product concentration is insignificant
    2. The substrate concentration is large and does not change significantly
    3. The free enzyme concentration is always in great excess to the concentration of the ES complex
  3. The catalytic rate constant Kcat is
    1. The rate at which substrate binds to an enzyme
    2. A measure of affinity of enzyme for substrate
    3. A constant evaluated by a Scatchard plot
    4. The forward rate constant for the rate limiting step of an enzyme

 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.)? 

Competitive. 

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….

  1. Cyanide
  2. Formaldehyde
  3. Formic Acid
  4. Dimethyl Ketone

Sulfa drugs are analogs of PABA. They function by…

  1. Competitively inhibiting biotin synthesis
  2. Uncompetitively inhibiting biotin synthesis
  3. Competitively inhibiting folic acid synthesis
  4. Irreversibly inhibiting folic acid synthesis
  5. Noncompetitively inhibiting peptidoglycan synthesis

Viagra works by…

  1. Stimulating NO synthesis
  2. Competitively inhibiting the breakdown of NO
  3. Irreversibly inhibiting a phosphodiesterase
  4. Stimulating cGMP synthesis
  5. Competitive inhibiting the hydrolysis of cGMP

In answer choice E, what type of enzyme classification catalyzes the hydrolysis of cGMP? 

Hydrolase

Irreversible Inhibitors.

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? 

Penicillin

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,

  1. Irreversibly inactivate acetylcholine esterase by forming a stable covalent bond with serine
  2. Inhibit acetylcholinesterase by transferring a phosphate group to the protein
  3. Are strong competitive (i.e. reversible) inhibitors of acetylcholinesterase
  4. Must first be hydrolyzed in order to be active

Bisubstrate Reactions

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.

  1. Single Displacement
    1. Random (Either substrate A or B can bind first; Either product P or Q can be released first)
    2. Ordered (Substrate A has to go before B binds; Product P has to be released before Q is)
  2. Double Displacement (Substrate A binds, part of substrate A is stuck to enzyme, changing the enzyme Product P is released, leaving the altered enzyme. E -> E’ (altered). Substrate B can only bind to E’

(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?

  1. ___Kinetic Analysis_____ – What do you uses to distinguish if mechanism is single or double displacement? Use a Line weaver burk plot. Parallel lines -> double displacement. Intersecting lines -> single displacement.

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

  1. ____Exchange Reactions_________ - Attempt reverse reaction in absence of one substrate. To distinguish what?

Btwn single and double displacement

Scatchard Plot  

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

  1. Stabilization of Transition State Intermediates
  2. Proximity and Orientation of Substrates
  3. Entropy Loss in ES formation
  4. Destabilization of ES due to:
    1. Geometric Strain (substrate may just not fit quite perfectly geometrically to enzyme since it’s ideal for the transition state)
    2. Electronic Strain (sometimes the substrate will have a (-) charge and the A.S. will have a (-) charge, causing an UNFAVORABLE charge-charge interaction. Or it can be +/+. Electronic strain does not ALWAYS happen.

It’s just one way that it can be destabilized)

  1. Desolvation of Substrate (Solvation -> stability. Desolvating -> destabilization. Desolvating because you are removing the susbstrate from the solvating environment)
  1. Formation of Unstable covalent intermediates
    1. With side chains of reactive standard amino acids
    2. With prosthetic groups, i.e., conezymes: pyridoxol phosphate (B6), thamine pyrophosphate, etc.
  2. General Acid/Base Catalysis
  3. Metal Ion Catalysis
  4. Stabilization of Transition State Intermediates.
    • Does the substrate or the transition state intermediate have better binding affinity to the enzyme? You can tell by the Km (of susbstrate) and Ki (of TSA/inhibitor) values. The Ki and Km can both stand for dissociation constant of their respective compound. The Ki is lower than that of the Km. That means the association for the inhibitor is higher; the binding affinity of the TSA is higher than that of the enzyme. The lower the Ki the better the inhibitor.

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. 

  • What is a TSA that prevents the influenza? What enzyme does it inhibit? What substrate does it block from binding? Tamiflu, inhibits Neuraminidase (major glycoprotein on virus membrane needed for replication), Substrate is Neuraminic acid (or Sialic Acid). So.. Tamiflu is an analog of the transition state when neuraminic acid reacts with the enzyme neuraminidase.

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).

  1. PROXIMITY AND ORIENTATION OF SUBSTRATE
    • What are the arrangements called that optimize proximity and orientation of substrate to enzyme?

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. 

  1. ENTROPY LOSS IN ES FORMATION
    • The free energy of ES is ___lower_________ (lower/higher) than that of E + S. 
    • Entropy loss will cause an _______increase______ (increase/decrease) in ΔG.
    • Why would entropy loss contribute to catalysis? You don’t want the ΔG of the ES complex to be too low and fall into an energy well, because getting to the next step, the transition state, would be more difficult. You want the ΔG of the ES to be lower than that of the E+S but still a little higher than E + P.
    • What happens when ES has too low of a ΔG? What does that do to the next activation barrier the reaction must cross to reach EP? See above. The next activation barrier is increased. The point of the enzyme is to make it easier for the reaction to happen. So if the ES free energy is too low, making the next activation hump too large, then the enzyme doesn’t really help.  

The dotted line is if the ES is never destabilized by anything and falls into the energy well, making it hard to overcome it.

  1. DESTABILIZATION OF ES DUE TO:
    • Will destabilization cause an increase or decrease in free energy? Increase
  2. FORMATION OF USNTABLE COVALENT INTERMEDIATE w/ reactive standard amino acids

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?

  • Trypsin – serine (B/C of –OH)
  • Papain – cysteine (-SH)
  • Acid Phosphatase – histidine (NH+) Aldolase – lysine (NH3+)

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?

Histidine

Which amino acid acts to stabilize orientation and structure of triad by H bonds and charge-charge interaction? 

Aspartic Acid/Aspartate

  1. GENERAL ACID/BASE CATALYSIS

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, 

ASPARTIC PROTEASES.

  • Do these proteases have a catalytic triad? No
  • How many reactive residues are in their active site? What are the residues? 2, 2 aspartates - Examples of aspartic proteases include PEPSIN, HIV PROTEASE.
  • PEPSIN is a sequence specific reagent (Chapter 4.7-5). What amino acids does it cleave after? Trp, Tyr, Phe, Leu

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 this an example of a serine protease or an aspartic protease? Aspartic protease - What is the importance of HIV protease in terms of HIV? What does it do?

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.  

  • Below is a picture of protease inhibitors. What structure do they look like? Proteins/polypeptides
  • In every inhibitor, there is a tetrahedral carbon in place of where a carbonyl carbon should be (carbonyl carbon of an polypeptide). Originally, the substrate would have a carbonyl carbon. However, the inhibitor has a tetrahedral carbon. What is the difference in geometric conformation between the two? What type of inhibitor would you call these? (Analogs of What?) First, trigonal planar. Then tetrahedral. These are transition state analogs because they mimic the structure of the TS. You can tell because it has the

geometry of the TS.

  1. Metal Ion Catalysis. Using Zn+2 (Or Copper, Iron, Magnesium)

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…

  1. Generate OH- at physiological pH
  2. Introduce electronic strain in the substrate
  3. Involve a coordination link between the metal ion and the transition state
  4. B and C
  5. All of the above

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. 

Enzymatic Cleavage.

  1. Trypsin; after; Arginine and Lysine (+ or basic A.A.)
  2. V8 Protease; After; Glu and Asp (- or acidic A.A.)
  3. Chymotrypsin; After; tryptophan, tyrosine, phenylalanine, Leucine (bulky aromatics plus leu)
  4. Pepsin; After; Tryptophan, Tyrosine, Phenylalanine, Leucine (bulky aromatics plus leu)
  5. Thermolysin; BEFORE; Ile, Val, Leu (nonpolar/hydrophobic)

Chemical Cleavage. 

  1. Cyanogen Bromide; after methionine
  2. Hydroxylamine; btwn Asn and Gly

3.BNPS-Skatole; after Trp

  1. o-iodosobenzoic Acid; 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? 

N-terminus

Chapter 6 Protein Structure.

Name your 4 levels of protein structure and what composes each.

  1. Primary – just your amino acid sequence
  2. Secondary – your repeating structures due to weak forces
  3. Supersecondary structures
  4. Domains
  5. Tertiary – your completely folded subunit
  6. Quaternary – Multisubunit protein. Like hemoglobin.

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?

Peptide Bond

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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