Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chemistry 431 - Fall 2008

CHEM 431	Exam II Study Guide	R. Paselk

2008 Version

Review

Study Guide I material, particularly as it pertains to enzymes and metabolism.What is an enzyme? (define) Turnover number. velocity.

Enzymes

Specificity

Lock and Key model and its failure. Induced fit model - explain. How do substrates bind? Chemical specificity. Why are enzymes big (<5% of surface is active site).

Zymogens: What are they? Why do they exist? (What enzymes commonly occur as zymogens?)

Catalysis

Know major types we discussed:

• general acid/base (as opposed to specific),
• covalent,
• proximity/orientation,
• distortion/transition state binding,
• transition state charge stabilization,
• metal ion.

What is meant by a "concerted" mechanism?

Be able to explain an enzyme mechanism in terms of the catalytic types we have discussed.

Be familiar with the mechanisms of lysozyme and the catalytic triad of the Serine Proteases we discussed in class.

• Given the reactions (substrate & product names and structures),
  • be able to show reasonable mechanisms including electron movements ("arrow pushing") for bond making/breaking,
  • be able to explain these mechanisms in catalytic terms.

Enzyme Kinetics

What are main assumptions in steady-state derivation of the Michaelis-Menten eqn?

Note the consequences of the M-M eqn at:

• [S] >> K_m,
• [S] << K_m,
• [S] = K_m.
• Be able to identify/explain reaction order at various [S].

Be able to interpret the Michaelis-Menten (v_i vs. [S]) and Lineweaver-Burke (double-reciprocal) plots for both uninhibited and inhibited reactions. Be able to find and/or show on/with both plots:

• V_max and apparent V_max
• K_m and apparent K_m.

Know the three type of classical, reversible Inhibition:

• competitive: model mechanism, kinetics, plots;
• non-competitive: model (mechanism), kinetics, plots;
• un-competitive: model (mechanism).

Be able to draw and interpret plots of:

• rate vs. temperature
• rate vs. pH.

Allosteric Enzymes:

• Define
• What do v_i vs. [S] plots look like for these enzymes?
• Cooperativity (what does this term mean in enymology),
  • Heterotropic.
  • Homotropic.
• Concerted (symmetry) model for homotropic allosteric enzymes.
  • Be able to explain model (shifting equil.) for substrates and effectors - correlate to kinetics.
• Sequential Model - explain.

Multisubstrate enzymes

Be able to draw and/or distinguish reaction mechanism diagrams for the multisubstrate mechanisms we looked at:

• Ping-Pong,
• Ordered Sequential
• Random Sequential

Be able to distinguish these mechanisms using product inhibition data and logic (e.g. "binding same enzyme form").

Carbohydrates

• What are the major functions for these molecules?
• Are there any patterns distinguishing fuel and structural forms of CHO's? Why?
• What are the common forms of fuel storage ("starchs")?
  • How do they differ?
  • What are advantages/disadvantages for each?
  • How do animals and plants differ? Why?
• Be able to describe how cellulose is able to accomplish its jobs and special properties it has.
• Memorize structures for: ribose, glucose, fructose, galactose, amylose, amylopectin, glycogen.

Lipids & Membranes

Lipids

• Fatty acids.
• Triacylglycerols (triglycerides).
• Phospholipids - what are the different families and how are they distinguished? What are the important/common R groups attached to the phosphate?
  • Micelle.
  • Bilayer.
• Memorize structures for: glycerol, choline, ethanolamine, stearic acid, oleic acid, palmitic acid.
  • Be able to draw phospholipids and/or triacylglycerols composed of any of the components above.

Membranes

• What are some membrane functions?
• Explain how chain length and saturation of fatty acid residues affect membrane fluidity.
  • Why does unsaturation affect fluidity?
    • Would you expect cis- and trans- versions of a given fatty acid to have the same fluidity? Why or why not?
  • Why does chain length affect fluidity?
• Why don't phopholipids "flip" in membranes?
• Does membrane lipid-bilayer composition differ inside vs. outside?
  • What distinguishes inner vs. outer bilayer lipids? (Both types of moleculaes and the geometrical rationale).
• How does cholesterol affect membrane fluidity?
• Lipid bilayers as barriers.
• Fluid Mosaic Model.
  • Lipid functions in membranes.
  • Protein functions in membranes.
• Membrane flow and membrane biogenesis.
  • How do proteins get into membranes?

Vitamins and Cofactors

What are vitamins?

• Why are they vitamins?
• Know relationship between vitamins and cofactors we have discussed.
• Niacin vs. NAD⁺ and NADP⁺
  • NAD⁺ = most common oxidation cofactor (catabolism)
  • NADP⁺ = most common reduction cofactor (anabolism)
• Riboflavin vs. FAD and FMN
  • stronger oxidizer then NAD⁺
• Pantothenate vs. Coenzyme A
  • carrier of acyl groups (activated)
• lipoate
  • linked to lysine to give lipoamide "arm."
• biotin
  • linked to lysine to give biotinamide "arm."
• In what portion (specifically) does the chemistry take place in each of these cofactors?
• What are the major metabolic functions of NAD⁺ and FAD?
• What distinguishes NAD⁺ from NADP⁺?
• What is the metabolic consequence of this difference?
  • What do we mean by metabolic compartmentation?
• What is the function of the "ADP" portion of NAD⁺, NADP⁺, and FAD?
  • What common theme connects NAD⁺, FAD, and Coenzyme A?
• What are the specific major metabolic functions of Coenzyme A, lipoic acid, and biotin?
  • What common themes connect these three cofactors?
  • How long is the acyl (pentanoate) plus lysine "arm" for lipoate and biotin (measure in "atoms")?
  • How does this compare to the "arm" in coenzyme A?
• What are the fat soluble vitamins and their major functions?
  • A (source of retinoate for rodopsin pigment in vision)
  • D (calcium uptake hormone)
  • E (anti-oxidant).

Metabolism

Overview of Metabolism: Catabolism. Anabolism. Autotroph. Phototroph. Chemotroph.

Thermodynamics

What is thermodynamics? What can it tell you about a process? Pathway independent.

• G What is G (in words)?
  • G° (definition)
  • G° ' (definition)
  • G = H- T S.
    • What does it indicate about energy availability?
    • What does G tell you about a system?
  • G = G° '+ RT lnQ.
  • G° ' = -RT lnK_eq.
• Know how to solve simple problems using thermo relationships.
  • How do you find G for a series of reactions? K_eq?
  • Driving reactions by coupling:
    • sequentially,
    • in parallel. (Note special function of enzymes in this case.)

Energy capture in catabolism

ATP is the common energy "currency" of metabolism.

• Why ATP?
  • Phosphoric acid anhydride bond is unstable to hydrolysis (thermodynamically unstable)
  • However, Phosphoric acid anhydride bond is not readily hydrolyzed (kinetically stable)
• Shifting Equilibria (G) with ATP.
• "Hi Energy".
  • What do we mean by "hi energy?" (The compound has a large negative G° ' of hydrolysis, where 'large' means greater than or equal to G° ' of ATP.)
• NAD(P)⁺ and Flavins as "universal" carriers for electrons.
  • Note chemically active vs. recognition portions of these molecules.

Know the Stages of Metabolism and what major processes occur in each stage.

• I
• II
• III
• IV

Energy regulation in catabolism:

• What is Energy charge?
  • How is it maintained?
  • Why is it of interest?
  • Be able to calculate E.C.

Memorize structures for: ATP, ADP, AMP, NAD⁺, NADH, NADP⁺, NADPH; phosphorylated derivatives of sugars (G-6-P, G-1-P, F-1,6-P etc.); glyceraldehyde, glycerate and phosphorylated derivatives; Pyruvate, lactate and PEP

Glycolysis

3 stages

• I, preparatory (Glu Æ F-1,6-bisP);
• II, oxidative phosphrylation (F-1,6-bisP Æ 2 x 3-PGA)
• III, energy generation (2 x 3-PGA Æ 2 x Pyr).
• Need to regenerate NAD⁺:
  • aerobic vs. anaerobic.
• What reactions are irreversible?
• Which enzymes are involved in control?
  • What are the regulatory effectors of these enzymes?
  • How are regulatory steps determined? (K_eq. vs. measured Q or deltaG).
• Which steps consume ATP?
• Which produce ATP?
• What cofactor is required by Kinases?
• Where are NAD⁺/NADH involved?
• Understand this pathway:
  • What kinds of chemical reactions are involved?
    • How are they catalyzed?
    • Correlation of enzyme names and chemical reactions.
  • Strategy of reactions in pathway:
    • Why split into three carbon pieces?
    • Why oxidize?
    • Why is PFK main regulatory step?
    • Why not first step of pathway?
  • Know cofactors used by various enzymes.
  • Be able to explain "hi energy" in various compounds we have discussed in this pathway.
  • Why is 2,3-BPG necessary?

Be familiar with detailed catalytic mechanisms for:

• Aldolase
  • Given the reaction (substrate & product names and structures),
    • be able to show reasonable mechanisms including electron movements ("arrow pushing") for bond making/breaking,
    • be able to explain their mechanisms in catalytic terms,
  • Be able to correlate the chemical mechanisms with kinetic mechanism.
• Glyceraldehyde-3-P DH
  • Given the reaction (substrate & product names and structures),
    • be able to show reasonable mechanisms including electron movements ("arrow pushing") for bond making/breaking,
    • be able to explain their mechanisms in catalytic terms,
  • Be able to correlate the chemical mechanisms with kinetic mechanism.

Glycolysis Review

Gluconeogenesis and Glycolytic Control

• Know the major branch points and entry points for sugars in carbohydrate metabolism (see metabolism handouts):
  • glycogen
  • glycerol-P
  • Pentose-P shunt
  • Kreb's Cycle
  • fructose
  • galactose
  • glycerol
• Know the "bypass" reactions of gluconeogenesis.
• How is glycolysis/gluconeogenesis linked to Kreb's Cycle?

Know how glycolysis/gluconeogenesis is controlled.

• Note the hierarchy of control.
• In what senses does PFK function as the main and most important control enzyme in glycolysis.
• Know what substances act as effectors for PFK and how they work.
  • ATP
  • AMP
    • Why is AMP used as the physiological regulator of PFK activity in advanced organisms (modern bacteria - eukaryotes)
• Know how PFK activity indirectly affects the activities of the other control enzymes of glycolysis/gluconeogenesis.
  • HK
  • PK
• Be able to discuss in detail the regulation of carbohydrate metabolism and, in particular, the regulation of PFK.

Gluconeogenesis Review

Pyruvate Metabolism

• What are various fates of pyruvate?
  • How is it catabolized?
  • How is pyruvate involved in filling the TCA cycle (anapleurosis)?
  • How is it involved in gluconeogenesis?
• Know overview of Pyruvate DH complex (as per Pathway Diagram)
  • List the cofactors used by this enzyme complex.
  • Mechanism of Pyruvate DH reaction, including cofactors (as per TPP attack & Lipoate attack we went over).
  • Given the reaction (substrate & product names and structures),
    • be able to show reasonable mechanisms including electron movements ("arrow pushing") for bond making/breaking,
    • be able to explain their mechanisms in catalytic terms,
  • How is complex regulated?
  • Covalent regulation.
• Know quaternary structure of mammalian complex (qualitative).

Memorize structures for the chemically active portions of: TPP, FAD, Lipoamide.

You may bring a data/information sheet to the exam, however you must not exceed one side of a single sheet of 8.5"x 11" paper (total surface of one-half of one standard sheet of paper) for this sheet! GOOD LUCK!

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Last modified 13 November 2008