Biochem Unit 2

Thermodynamics of Transport for Charged Species

Where C is the concentration,

Z is the on the ion being transported

F is the Faraday constant (96.5 kJ/volt-mol) and psi is the electrical potential of the solution

Delta psi is the voltage difference across the membrane

Passive Diffusion

For uncharged molecules, passive diffusion is an entropic process. If C2 is less than C1 , then delta G is negative, and the process occurs

facilitated diffusion

• Transported species moves down electrochemical gradient

• Usually faster than passive processes

• Membrane protein or other “carrier” involved

Passive vs facilitated diffusion

For passive diffusion, the rate is proportional to concentration of diffusing species

For facilitated diffusion, the rate of transport is saturable

Example of Facilitated Diffusion

Glucose transporter in erythrocytes

cytochalasin B

a inhibitor from a fungus, that binds competitively to the glucose transporter and inhibits it

Active Transport

Energy of ATP hydrolysis used to do work of transport

Na +, K+ -ATPase

• Pumps Na + out of cells, K+ into cells (2K+/3Na +)

• Ion gradients important in nerve transmission, and in “cotransport” of other species

• Two subunits

• Phosphorylation/dephosphorylation and two protein conformations (conformations change after binding to ATP) involved

• Specific inhibitor—cardiac glycosides

This process is

charge neutral due to H+ and 2K+ coming in

Ca 2+ -ATPase

• Ca 2+ is a cellular “second messenger” in virtually all cells

• Normally Ca 2+ is kept low by pumping it into cellular vesicles called the sarcoplasmic reticulum.

• Pumping is by an ATP-driven, Ca 2+ -ATPase

• Important in lowering concentration of Ca 2+ released after muscle contraction

• Some protein homology to Na +, K+ -ATPase, both in structure and in mechanism

H+ -ATPases

• Gastric H +, K+ -ATPase

  • K+ , Cl - symport makes it an HCl pump

• Vacuoles and Osteoclast

• Mitochondrial and Chloroplast ATPases

  • Role of these pumps is to use proton gradient to drive synthesis of ATP rather than ATP hydrolysis to drive pumping of protons

Osteoclast Proton pump

The osteoclast pumps out protons right by the bone, solubilizing it, for providing Ca+2 elsewhere in the body

Ion Gradient Driven Active Transport/Secondary Active Transport

cotransport ions go down their electrochemical gradient and other molecules (glucose or amino acids) goes up their electrochemical gradient

Symport

Substance transported in same direction of ion

Antiport

Substance transported in opposite direction of ion

Light Driven Transport

Bacteriorhodopsin

• a major membrane protein of Halobacterium halobium, forming purple patches in membrane

• Retinal bound as Schiff base to lysine residue of bacteriorhodopsin

• Light absorption promotes trans to cis isomerization

  of the retinal

• Conformational changes during isomerization results in pumping protons out of cell

Porins

• Pore forming proteins

• Relatively non-specific

• Outer membranes of bacteria and mitochondria

Gap Junctions

Forms connections between cells

(the one shown in this picture is a homohexamer)

Ionophores

• Mobile carrier

  • Cyclic peptide, valinomycin, is an example

• Channel forming peptides

  • Gramicidin as an example

cyclic ionophore, valinomycin

specific for K+1

prefix m

x 10^-3

prefix µ

x 10^-6

prefix n

x 10^-9

monomer of nucleic acids

nucleotides

Nucleotides composition

• Heterocyclic Base

• Pentose

• Phosphate

pyrimidine ring

Note the numbering system is different for the two types of ring structures

purine ring

Note the numbering system is different for the two types of ring structures

cytosine

uracil

thymine

carbonyl is more stable

adenine

guanine

carbonyl is more stable

syn conformation

anti conformation

useful property in measuring quantities of nucleic acid in a sample

Both pyrimidines and purines have strong absorbance in the UV spectrum around 260 nm

Nucleosides

beta glycosidic linkage at N-1 of pyrimidine

and N-9 of purine

Nucleoside Nomenclature for pyrimidine

add -idine to the root name

Nucleoside Nomenclature for purine

add -osine to the root name

expect hypoxanthine

Nucleosides of deoxyribose are deoxyribonucleosides and are prefixed by

deoxy

expect for thymidine (only found in DNA anyway)

Nucleotides nomenclature

Add –ylic acid to base stem

Nucleotides

• Nucleotides are phosphate esters of nucleosides

• Named as “nucleoside-X’-phosphate” where X’ is the ribose position to which the phosphate is attached

• Example: adenosine-5’-monophosphate

Triphosphates are

energy intermediates

– ATP major energy currency

– GTP involved in driving protein synthesis

What kind of chemical bond links nucleotides together?

phosphodiester

How to read DNA

READ IT 5’ to 3’

How many H-bonds are there per base pair?

2

How many H- bonds are there per base pair?

3

Nature of DNA Helix

• Antiparallel strands

• Ribose phosphate chain on outside

• Bases stacked in middle like steps in a spiral staircase

• Complementary strands provide possible mechanism for replication

Messenger RNA

• “Transcription” product of DNA

• Carries sequence information for proteins

• Prokaryote mRNA may code for multiple proteins

• Eukaryote mRNA codes for single protein, but code (“exon”) might be separated by non-coding sequence (“introns”)

Ribosomal RNA

• “Scaffold” for proteins involved in protein synthesis

• RNA has catalytic activity as the “peptidyl transferase” which forms the peptide bond

• Prokaryotes and eukaryotes have slightly different ribosomal structures

• Ribosomal RNA contains some modified nucleosides

Transfer RNA

• Small molecules—73-94 residues

• Covalently carries an amino acid for protein synthesis

• One or more t-RNA’s for each amino acid

• “Anti-codon” in t-RNA recognizes the nucleotide “code word” in m-RNA

• 3’-Terminal sequence always CCA

• Amino acid attached to 2’ or 3’ of 3’-terminal A

• Many modified bases

Small Nuclear RNA’s

• Found in eukaryotic cells, principally in the nucleus

• Similar in size to t-RNA

• Complexed with proteins in small nuclear ribonucleoprotein particles or snRNPs

• Involved in processing eukaryotic transcripts into m-RNA

RNA hydrolysis

RNA can be hydrolyzed by base because of the 2’-OH group. Mixture of 2’ and 3’ nucleotides produced

DNA hydrolysis

DNA can be hydrolyzed by an acid, by only the glycosidic bonds of the purines are broken (apurinic acid)

Exonucleases

hydrolyze terminal nucleotides

Endonucleases

hydrolyze in middle of chain. Some have specificity as to the base at which hydrolysis occurs

a type nucleases

cleaves the 3’ phosphate bond

Produces 5’-phosphate products

b type nucleases

cleaves the 5’ phosphate bond

Restriction Endonucleases

• Enzymes of bacteria that hydrolyze “foreign” DNA

• Name based on “restricted growth” of bacterial viruses

• Enzymes specific for a short sequence of nucleotides (4-8 bases in length)

• Methylation of the same sequence protects “self” DNA from hydrolysis

Specificity of Restriction Endonucleases

4-base sequence occurs randomly every 4^4

bases, or every 256 bases

6-base sequence occurs randomly every 4^6

bases, or every 4096 bases

8-base sequence occurs randomly every 4^8

bases, or every 65,536 bases

palindromes

Many target sequences, called “restriction

sites”

Cleavage of palindromic sites leave single stranded “sticky ends”, either 5’ or 3’

Restriction Mapping

• Samples of DNA cut with a particular restriction enzyme yield a set of characteristic polynucleotides, separable electrophoresis according to size.

• Each enzyme produces its own characteristic set of sized fragments

• Fragments can be reassembled as in a jigsaw puzzle to produce a “restriction map”

Electrophoresis of DNA molecules

separates them according to size, the largest being retarded in their migration through the gel pores while the smallest move relatively unhindered

B-form of DNA

- Right-handed

~10 bp per turn

most common (no space for water in the middle)

-anti conformation

A-DNA

- -Right-handed

~11 bp per turn

-evenly spaced (and room for water)

-anti conformation

Z-DNA

-Left-handed

-CG base pair

-12 bp per turn (less space for water)

syn conformation

how many hydrogen bonds in AT base pairs?

2

how many hydrogen bonds in CG base pairs?

3

glycosidic bond

bond between sugar and nitrogenous base

Methylation of C

promotes B DNA to Z DNA switch

Increase of CG content and increase of ionic strength equals

increase in DNA stability

Reannealing of DNA

• Reannealing is the term given to the

renaturation—the reformation of the helix

• Temperature must be lowered slowly for proper

nucleation to occur

• Renaturation is a second order process, so the rate depends on concentration of complementary base

sequences

Chromatin

The nucleoprotein complex of DNA found within the eukaryotic cell nucleus

histones

pairs of histones (chromatin is wrapped around histones) aggregate to form octameric nucleosomes

Transfer RNA Structure

• Secondary structure shows a “cloverleaf”

pattern. All tRNA’s are similar.

• All end in CCA- 3’ , with amino acid attached at

the 3’-OH

• Lots of unusual modified bases.

• Tertiary structure is L-shaped

– Lots of “noncanonical H-bonding.

Ribosomal RNA Structure

A great deal of intra-strand sequence complementarity

Enzymes are intrinsically different from ordinary chemical catalysts

1) Higher reaction rate reactions must proceed on the time scale of living systems

2) Milder reaction conditions reactions must take place in an aqueous environment

3) Greater specificity many biological metabolites appear similar in structure

4) Capacity for regulation ability to sense moment to moment metabolic needs

Rate Enhancment

kcat /knon

t 1/2 (half life)

(ln 2)/kobs

Lock-and-Key Models

Molecules, like enzymes and substrates, need to fit together like a lock and key for things to work correctly

Induced Fit Theory

So, the key idea here is that the protein only reacts with the right substrate by changing its shape in a specific way

substrate

The biological molecule that gets chemically transformed by an enzyme

active site

the unique three-dimensional pocket where the substrate binds and where chemistry takes place

cofactor

If an enzyme makes use of any non-proteinogenic compound to facilitate catalysis (vitamins)

inhibitor

Any molecule that interferes with enzymatic catalysis by slowing any step of the chemical transformation

Ligase

1) forming C-O bonds

2) forming C-S bonds

3) forming C-N bonds

4) forming C-C bonds

usually powered by ATP hydrolysis

Oxidoreductases

These enzymes conduct oxidation-reduction reactions on biomolecules using a variety of oxidizing/reducing agents

Transferases

These enzymes catalyze the transfer of many functional groups including phosphoryl, methyl, acyl, glycosyl, etc

Hydrolase

These enzymes catalyze the hydrolysis of various chemical bonds within biomolecules

Specific Example: BamHI restriction endonuclease Phosphodiester bond hydrolysis

Lyases

These enzymes cleave chemical bonds, often via addition across a double bond

Isomerase

These enzymes catalyze the interconversion of structural isomers

prosthetic group

an organic molecule thats is a helper and is tightly bound, or covalently attached to the enzyme

Holoenzyme

enzyme plus prosthetic group

Apoenzyme

enzyme lacking it’s prosthetic group

keq (rate constant)

kdec

k obs

Keq * Kdec

Kcat and knon

are types of kobs

enzymes and kinetics

They speed up the rate of reactions, without affecting the thermodynamics of the reaction

They are able to do this by lowering the Activation Energy