Chemistry 20284 - Physiological Chemistry
Spring 1997 - Exam #2 Review

Contents

• Chapter 5: The Behavior of Proteins: Enzymes
• Chapter 6: Nucleic Acid Structure
• Chapter 7: Nucleic Acid Biotechnology
• Chapter 8: Lipids and Membranes

Chapter 5: The Behavior of Proteins

Definitions

Activation Energy (E_a or DG^¹)

Energy required to initiate a chemical reaction.

Active Site

Physical location on a protein where enzyme-catalyzed reaction occurs.

Allosteric Enzyme

Behavior of enzyme changes as a result of substrate binding.

Catalyst

A chemical compound that lowers the activation energy of a chemical reaction. Although DG does not change, lowering E_a increases the reaction rate.

Enzyme

Globular protein which acts as a catalyst for biological reactions.

Feedback Inhibition

Sequence of enzymatic reactions produces a substance that inhibits further reaction.

Intermediate

Substance produced in one step of a reaction mechanism, but consumed in a subsequent step. Not present at either beginning or end of reaction.

Kinetics

Study of rates of chemical reactions.

Mechanism

Sequence of steps required to complete a reaction.

Substrate

Reacting substance that binds to active site of an enzyme.

Thermodynamics

Study of energy changes of chemical reactions.

Zymogen

An inactive precursor that can "easily" be converted to an active form.

Rate Laws

General Rate Equation

Rate = k [A]^a [B]^b ...

Reaction order = a + b + ...
Order of reaction with respect to [A] = a

Enzymatic Reactions

Lock-and-Key Mechanism

Enzyme assumed to be rigid. Substrate must have the exact "right" geometry to fit in active site.

Induced Fit Mechanism

Enzyme assumed to be flexible. Geometry of enzyme can change to accommodate substrate.

Competitive Inhibition

Inhibitor binds at active site of enzyme.

Non-competitive Inhibition

Inhibitor binds at site other than active site, which changes enzyme-substrate affinity.

Michaelis-Menton model for Enzyme Kinetics

Plot of reaction rate (y-axis) vs. concentration of substrate (x-axis) is hyperbolic. At low [S], first order behavior. At high [S], zeroth order behavior.  Reaction kinetics can be explained using equations derived from the Michaelis-Menton model:

V

Reaction rate. At high concentration of substrate, approximately V_max. At low concentrations of substrate, follows first-order kinetics.

V_max

Maximum rate of reaction.

K_M

Michaelis constant. Large values indicate substrate bonds weakly to enzyme.

[S]

Concentration of substrate.

Kinetic Analysis: Lineweaver-Burk Plot

Plot of V^-1 (y-axis) vs. [S]^-1 (x-axis) should be linear with:
Slope = K_M / V_max
Intercept = 1 / V_max

Chapter 6: Nucleic Acid Structure

Nucleic Acids

Nucleoside

Consists of a base and a sugar.

Nucleotide

Consists of a base, a sugar, and a phosphate group.

Polynucleotide (nucleic acid)

Polymer of nucleotides linked by phosphodiester bonds.

Nucleic Acid Bases

Heterocyclic compounds based on either purine or pyrimidine. Contain both H-bond donor and acceptor sites. See p 181 of text for structures.

Sugars

Nucleic acids use either ribose or deoxyribose. Both are five-carbon sugars with a five-membered ring containing 4 C and 1 O atom.

Glycosidic Bond

N-H group of base reacts with C₁-OH group of sugar to form a covalent C-N single bond. See Figure 6.4 (p 183) in text.

Phosphate Group

PO₄^3- reacts with C₅-OH group of nucleoside sugar to form a nucleotide. Phosphate groups can further react with the C₃-OH group of other nucleotides to form large chains linked by phosphodiester bonds. Phosphate group maintains a negative charge.

Structure of DNA (deoxyribonucleic acid)

The primary structure of polynucleotides describes the sequence of nucleotides present. The secondary structure describes the "local" 3-dimensional structure. For DNA, the secondary structure is a double helix, which is driven by H-bonding between complementary base pairs (A-T and C-G). The tertiary structure can be viewed as the "long-range" 3-dimensional shape of the molecule. For simple (prokaryotic) cells, the tertiary structure can be as simple as a circle. Complications include "supercoiling", which is essentially additional twists in this ring. An example of this might look like a "figure-8". For complex (eukaryotic) cells, the DNA tends to wrap around positively-charged, basic proteins call histones to form chromatin.

Hydrogen Bonds

Adenine (A) and Thymine (T) pairs form two H-bonds in DNA, while cytosine (C) and guanine (G) pairs form three H-bonds. Increasing the relative number of GC pairs (relative to A=T) raises the "melting point" of the DNA. See also the "handout" on H-bonding in DNA.

Double Helix

See p 190 of text for structure. Double helix found only in DNA, not RNA. Negative charge of phosphate groups located on "outside" of helix. Grooves allow substrates to "bond" to surface and access interior base pairs.

RNA (ribonucleic acid)

(Select text or pictures in above diagram
for additional information)

tRNA (transfer)

Carries amino acids to site of protein synthesis. Typically small, with significant H-bonding.

rRNA (ribosomal)

Combines with proteins to form ribosomes, which are the sites of protein synthesis. Typically large, with significant H-bonding.

mRNA (messenger)

"Translates" base sequence from DNA to ribosome. Ribosomes then use this information to determine sequence of amino acids in protein. Variable size, typically with very little H-bonding.

Structure

RNA is a single strand that winds back on itself to form "clover-leaf" pattern. Contains regions where H-bonding is important and open regions where it is not.

Restriction Enzymes

These enzymes locate specific base sequences in DNA and cleave both strands. Sequence recognized on one strand is exact reverse of sequence on second strand (palindrome), and cleavage occurs between same base pairs on both strands. If breaks occur at different positions on both strands, "sticky ends" produced. If DNA from two different sources cleaved with the same restriction enzyme and then recombined using DNA ligase, new (recombinant) DNA is formed. See Figures 6.22 and 6.23 of text (pp 204 & 206).

For example, a restriction enzyme might recognize the following nucleotide sequences and cause cleavage at the positions indicated to yield the products shown below.

Chapter 7: Nucleic Acid Biotechnology

Separation and Detection Methods

Gel Electrophoresis

Method for separation of DNA or RNA fragments based on size. All fragments move toward the positively-charged electrode, with the smaller fragments moving faster.

Autoradiography

Incorporation of radioactive ³²P makes fragment visible to X-ray film.

Fluorescence

Fluorescent fragment (label) bonded to DNA. More sensitive method.

Determining Base Sequences in Nucleic Acids

Ribose or deoxyribose sugar portion in small percentage of nucleotides replaced with dideoxyribose. This interrupts chain growth of growing cDNA (complementary) strand because the C₃' position no longer has the -OH group required to react with the phosphate on the "next" nucleotide. Fragments analyzed by gel electrophoresis. (See Figure 7.3, p 217).

Recombinant DNA

Virus or bacteria used as carrier (vector). DNA of vector and DNA of interest cleaved by same restriction enzyme, mixed, and reformed by DNA ligase. Recombinant DNA re-introduced in vector and grown in thin layers to produce large number of clones. If fragments of entire DNA of organism used, resulting clones are a DNA library. See Figures 7.11 (p 224) and 7.13 (p 226) for examples of how these libraries are analyzed.

The polymerase chain reaction is an alternative method for creating clones. This method is faster, and can be used to grow DNA under much less ideal conditions.

Gene Mapping

Classically done by repeatedly comparing DNA strands broken at different points (see Figure 7.15, p 229). More recent efforts make use of recombinant DNA techniques (restriction enzymes, probes, and electrophoresis). Idea is to determine how close (or far apart) select genes are from each other, and use this information to "piece together" order of genes on DNA strand. See also the worked out example: Use of Restriction Enzymes for Gene Mapping.

Chapter 8: Lipids and Membranes

Definitions

Lipids

An assortment of predominantly non-polar compounds that contain a limited number of polar groups. Only marginally water-soluble.

Membranes

Formed by lipid bilayers. Polar ends on surfaces, non-polar portions inside. Bulkier groups tend to be on outermost layer. Steroids and saturated chains raise melting temperature, while unsaturated chains lower melting temperature.

Membrane Proteins

Classified as integral (inside) or peripheral (outside). Have key roles in catalysis and transport.

Condensation

Fatty acids + glycerol ¾® triglyceride + water

Hydrolysis

Triglyceride + water ¾® fatty acids
(If base used, reaction is referred to as saponification)

Common Classes of Lipids

fatty acids	triacylglycerides	phosphoacylglycerides	steroids

(Fatty acid chain lengths typically even, and run from ~12 to ~20.)

Transport of Compounds through Membranes

Passive

Movement of molecules/ions from regions of high concentration to lower concentration.

Simple diffusion

Molecules move directly through membrane wall or a hole (channel).

Facilitated diffusion

Molecule bonds to carrier protein before transport.

Active

Movement of molecules/ions against concentration gradient. Requires energy and a channel protein.

Section 8.7 of text on neuromuscular junctions is a good example of membrane properties.
Includes a discussion of Voltage-gated and Ligand-gated channels.

Lipid-Soluble Vitamins

Vitamin A

cis/trans photo-induced isomerism plays a key role in vision.

Vitamin D

Important for calcium and phosphorous regulation. Derived from cholesterol.

Vitamin E

Anti-oxidant and free-radical scavenger.

Vitamin K

Plays a key role in blood clotting.