biochemistry lectures

proteins

nucleic acids

metabolism

CHEMISTRY 251/Fall 2012Instructor: Prof. Barry S. Cooperman 358 Chemistry cooprman@pobox.upenn.eduLecture: MWF 9 am in room Chem 102Prerequisite: Chem 102; Chem 241 (can be taken concurrently)Required Textbook: Fundamentals of Biochemistry. 4th edition (2012) - Voet, Voet, and Pratt Office hours: arranged by email

Lecture schedule (approximate) Sept 5 – Introduction - Chapters 1, 2Sept 7 - 12 Amino acids and protein primary structure Chapters 4, 5Sept 14 – 21 Protein structure and function Chapters 5, 6Sept 24 – 26 Enzymes, Chapters 11 and 12Sept 28, Oct 1 – Other Protein Functions chapters 7, 28Oct 3 Exam 1Oct 5-12 Nucleotides, Nucleic Acids and Nucleic Acid Structure - Chapters 3, 24Oct 15 - 19 DNA Replication - Chapter 25Oct 22 – 26 DNA Transcription Chapter 26 Oct 29 – Nov. 2 Translation Chapter 27Nov 5 Regulation of Gene Expression, small RNAs Chapter 28 Nov 7 Exam 2 Nov 9 Metabolism (general) Chapter 14Nov 12 – 16 Carbohydrates, Glucose and Glycogen Metabolism - Chapters 8, 15 & 16Nov 19, 21 Lipids, membranes, and lipid metabolism Chapters 9, 10, 20Nov 26 – Nov 30 - Citric Acid Cycle, Electron Transport, Oxidative Phosphorylation - Chapters 17 & 18Dec 3 - Exam 3Dec 5, 7 - Amino Acid Metabolism, Mammalian Metabolism – Chapters 13, 21, 22

-weekly recitation –voluntary – times and locations to be announced-There will be three hourly exams and one final. -Grades will be based on 100 points for each hourly exam and 150 points for the final. -Lecture notes and power point files will be posted on Blackboard page for Chem 251-Make-up lectures: I will be unable to lecture on Wed Sept 12 and Wed Sept 26. To make-up for the time lost, the following lectures will begin at 8:30 am instead of 9:00 am: Mon Sept 10, Fri Sept 14, Mon Sept 24, Fri Sept 28.

How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?

Erwin Schrödinger, What is Life?

MetabolismSum of chemical and physical reactions carried out by cell

Catabolism - breakdown of nutrients and cell components to generate energy and common precursors (building blocks); reactions are generally exergonic oxidations:- GAnabolism - synthesis of biomolecules - endergonic :+ G

Obligate aerobes - use O2 for oxdtn; animalsObligate anaerobes - O2 toxic; use other oxidantse.g., sulfate, nitrate -some bacteriaFacultative anaerobes - can use O2 or other oxidants - e.g., E. coli

Energy storage -High energy phosphate bonds; ATP: thioestersReduced cofactors: NADH, NADPH, FADH2

Generated by catabolic processes;Consumed by anabolic processesIntercoverted by oxidative phosphorylation

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The Central Dogma

General structural formula for -amino acids

Zwitterionic form

Pag

e 66

Cystine

Greek letter convention for naming side chains

Titration curve of glycine

Condensation of two -amino acids to form a dipeptide

The tetrapeptide Ala-Tyr-Asp-Gly

Note that within a peptide, the -amino and -carboxyl ionizationsare suppressed, except for the amino terminus and the carboxyl terminus. However, side-chain ionizations are maintained.

Aminoterminus

Carboxyl terminus

Side chain ionization

Ionizable side chains

Isoelectric Point of a Protein - no net charge -net charge (pH 7) = Arg + Lys + fHHis) - Asp + Glu + fCCys)-example shown is basic protein; (Arg + Lys) > (Glu + Asp + Cys)-so reaching isoelectric point requires deprotonation of some Lys residues (pKa 10.5)-for acidic protein: (Arg + Lys + His) < (Glu + Asp) - so reaching isoelectric point requires His protonation (pKa 6), and possibly some Glu or Asp protonation (pKa 4.5)

Convention for naming the enantiomers of amino acids

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A tetrahedral C with four different substituentsis chiral; has a nonsuperposable mirror image

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Modified amino acid residues occurring in proteins

Biologically Active Compounds derived from Amino Acids

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Insulin

Intra- and inter-chain disulfides (cystines)

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Sizing ProteinsMALDI-TOF MASSSPECTROSCOPYMANN ET AL.

Annu. Rev. Biochem. 2001. 70:437–73

Figure 1 Schematic of MALDI process and instrument. (A) A sample cocrystallized withthe matrix is irradiated by a laser beam, leading to sublimation and ionization of peptides. (B) About 100–500 ns after the laser pulse, a strong acceleration field is switched on (delayed extraction), which imparts a fixed kinetic energy to the ions produced by the MALDI process. These ions travel down a flight tube and are turned around in an ion mirror, or reflector, to correct for initial energy differences. The mass-to-charge ratio is related to the time it takes an ion to reach the detector; the lighter ions arrive first. The ions are detected by a channeltron electron multiplier.

MALDI-TOFMS of ribonucleotide reductase large subunit.Secondary peaks correspond

to a doubly charged mass.P. Foo, 2002

Myoglobin: Shown are mass/chargeratios

Positive charges due to Arg and Lys residues

Sizing proteins by Electrospray Ionization (ESI) Mass spectrometry

Fragmentation of Protein Into Peptide Fragments: Tandem Mass Spectrometry

Gel electrophores

is

Sucrose density gradient

ultra

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s =v

ω 2r=

M(1− v_

ρ )D

RT

D M-1/3; s M2/3

Shape dependence}

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RS-SR' + R"SH RSH +R'S-SR"

R'S-SR" + R"SH R'SH +R"S-SR"

RS-SR' = polypeptide; R"SH = 2-mercaptoethanol1

2RSH + ICH2CO2

- RSCH2CO2- + I- + H+

1

2

If no disulfides, start here

Protein Sequencing-1

Disulfide reduction

Protein Sequencing-2

Chemical cleavage - CNBr

At Mets

Process can be repeated on new N-terminus; can be repeated up to ~50 cycles

N-terminal Sequencing:The Edman Degradation

typical outputfrom amino acidanalysis

Sequencing with overlapping fragments

Tandem Mass Spectrometry for Sequencing

1) Proteinase digestion of protein

2) ESI-MS to generate peptide ions from digest (MS-1)

3) Collision cell to generate fragments of each ion

4) MS analysis of fragment ions (MS-2)

5) In this example, Peptide P3 fragments into F1 - F5 which are overlapping, allowing sequence to be deduced.