CHEMISTRY 251/Fall 2012 Instructor: Prof. Barry S. Cooperman 358 Chemistry cooprman@pobox.upenn.

edu Lecture: MWF 9 am in room Chem 102 Prerequisite: 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, 2 Sept 7 - 12 Amino acids and protein primary structure Chapters 4, 5 Sept 14 21 Protein structure and function Chapters 5, 6 proteins Sept 24 26 Enzymes, Chapters 11 and 12 Sept 28, Oct 1 Other Protein Functions chapters 7, 28 Oct 3 Exam 1 Oct 5-12 Nucleotides, Nucleic Acids and Nucleic Acid Structure - Chapters 3, 24 nucleic acids Oct 15 - 19 DNA Replication - Chapter 25 Oct 22 26 DNA Transcription Chapter 26 Oct 29 Nov. 2 Translation Chapter 27 Nov 5 Regulation of Gene Expression, small RNAs Chapter 28 Nov 7 Exam 2 Nov 9 Metabolism (general) Chapter 14 metabolism Nov 12 16 Carbohydrates, Glucose and Glycogen Metabolism - Chapters 8, 15 & 16 Nov 19, 21 Lipids, membranes, and lipid metabolism Chapters 9, 10, 20 Nov 26 Nov 30 - Citric Acid Cycle, Electron Transport, Oxidative Phosphorylation - Chapters 17 & 18 Dec 3 - Exam 3 Dec 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 Schrdinger, What is Life?

Metabolism
Sum 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:- DG Anabolism - synthesis of biomolecules - endergonic :+ DG Obligate aerobes - use O2 for oxdtn; animals Obligate anaerobes - O2 toxic; use other oxidants e.g., sulfate, nitrate -some bacteria Facultative anaerobes - can use O2 or other oxidants - e.g., E. coli Energy storage High energy phosphate bonds; ATP: thioesters Reduced cofactors: NADH, NADPH, FADH2 Generated by catabolic processes; Consumed by anabolic processes Intercoverted by oxidative phosphorylation
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The Central Dogma

General structural formula for a-amino acids

Zwitterionic form

Page 66

Cystine

Greek letter convention for naming side chains

Titration curve of glycine

Condensation of two a-amino acids to form a dipeptide

The tetrapeptide Ala-Tyr-Asp-Gly

Side chain ionization

Amino terminus

Carboxyl terminus

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

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 substituents is chiral; has a nonsuperposable mirror image

Modified amino acid residues occurring in proteins

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Biologically Active Compounds derived from Amino Acids

Insulin

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Intra- and inter-chain disulfides (cystines)

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Sizing Proteins MALDI-TOF MASS SPECTROSCOPY

MANN ET AL. Annu. Rev. Biochem. 2001. 70:43773

Figure 1 Schematic of MALDI process and instrument. (A) A sample cocrystallized with the matrix is irradiated by a laser beam, leading to sublimation and ionization of peptides. (B) About 100500 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

Sizing proteins by Electrospray Ionization (ESI) Mass spectrometry

Positive charges due to Arg and Lys residues

Myoglobin: Shown Fragmentation of Protein Into Peptide are mass/charge Fragments: Tandem Mass Spectrometry ratios

Gel electrophoresis

Sucrose density gradient

ultra

2r

M(1 v )D RT

D a M-1/3; s a M2/3

Shape dependence

Protein Sequencing-1
Disulfide reduction

1
If no disulfides, start here
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RS-SR' = polypeptide; R"SH = 2-mercaptoethanol RS-SR' + R"SH R'S-SR" + R"SH RSH +R'S-SR" R'SH +R"S-SR"

2
RSH + ICH2CO2RSCH2CO2- + I- + H+

Protein Sequencing-2

Chemical cleavage - CNBr

At Mets

N-terminal Sequencing: The Edman Degradation

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

typical output from amino acid analysis

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.