Biochemistry Chem-641 (Fall 2007 – part II)
Please complete teaching evaluation for CHEM641 http://www.udel.edu/course-evals
There are two pages – the first is for Dr. Bahnson the second for Dr. Thorpe
< <<
<<< <<<<
>>>> >>> >> >
HELP SESSION ON
SUNDAY EVENING
7 pm - 8 pm >>>
Dr. Bahnson
8 pm – 9 pm >>> Dr. Thorpe
You are welcome to come to either or both
WHERE?: SMITH HALL 140
=============================================================
DR. BAHNSON AND CT HAVE AGREED THAT YOU MAY BRING IN A SINGLE PIECE OF
8.5 X 11 INCH PAPER WITH ANYTHING WRITTEN OR PRINTED OR PAINTED OR PHOTOCOPIED
ON ONE OR BOTH SIDES TO THE FINAL EXAM …
Of course this is optional – some find this helpful … others do not. Your choice.
Remember ONLY one sheet of 8.5 x 11 inch paper – SO YOU WILL HAVE TO
DECIDE the coverage between the two halves of the course.
========================================================
For CT part … PLEASE
NOTE … additional exam material:
CHEM641_final exam +
key (an example of the 75 point format – from 2005)
>>>This is
just the Thorpe part of final
EXAM: chem641_05F_final_CT.pdf
EXAM KEY: chem641_05F_final_CT_KEY.pdf
===============================================================
For the FINAL exam next Wednesday,
about 60 points of the 75 total points of the Thorpe part will feature “new
material” (that was not covered on the second exam).
Metabolism will feature
prominently in the exam.
=================================================
Instructor: Colin Thorpe
204
Drake Hall
Phone:
831-2689
EMail: cthorpe@udel.edu
Office hours: Tue 10:45-11:45 am, Thu 3:00-4:00 pm, or
by appointment
====================================================================
Biochemistry is bioCHEMISTRY
EXAM PDFs
CHEM 641 2nd exam 2005 EXAM…Chem641F05.pdf
CHEM 641 2nd exam 2005 EXAM KEY … CHEM641_PDF_07\CHEM
641 Second Exam F05.pdf
CHEM 527 2nd hourly 2006F Exam CHEM641_PDF_07\exam2_Chem527_06F.pdf
CHEM 527 2nd hourly 2006F KEY (the correct one – sorry) CHEM 527 Second Ex_Key F06.pdf
CHEM 527F Final 2006F Exam CHEM641_PDF_07\chem527_Final_ex_F2006.pdf
CHEM 527F Final 2006F Key CHEM641_PDF_07\CHEM527_06F_FinalKEY.pdf
=================================================
>>KEY to second exam:
KEY_2ndexamCHEM641_07F.pdf
Please consult key before raising grading issues
As always, we will promptly address grading errors.
Please note there was a significant typo in ONE part of a question that
was noticed by one of you (thanks!). I
did not announce this mistake because about 20 people had already completed
their exams before the error was apparent.
I have ignored this part of the question in grading.
Average 68.5% (higher than usual – as someone observed the exam was
“predictable”)
LOW 26%
HIGH 99%
=============================================================================
ANNOUNCEMENTS
Please note the "no make
up" exam policy as set out in the syllabus.
Office Hours:
Office hours: Tue 10:45-11:45a, Thu 3:00-4:00p, or by
appointment
THE EXAM … Saturday
10 - !2 noon 100 KRB
You should bring a calculator.
Two review sessions ….
note time changes
Thur 15th
7:30 PM to 9 PM 205 GORE
Fri 16th 7PM – 9 PM 205 GORE
At that time I will answer questions – so bring them
along … no lecture/presentation.
You are welcome to come at any time – but if come
later and ask a question that has already been covered
that session – we may defer revisiting it to the end of the period.
EXAM COVERAGE – UP TO AND INCLUDING ALL OF GLYCOLYSIS
(BUT NOT TCA)
I will not deliberately test on material specifically
covered by Dr. B.
As promised - there will be a glycolysis
handout provided on the exam.
Exams come principally from lecture and not from the
book.
There was a
question in class about what chapters …. some readings
that were suggested in the first part of the course – they are scattered in the lecture synopses and
collected here.
please
review Chapter 6 (we will not cover pp. 219-233 directly here; I understand
that Dr. B has dealt with some of that material)
Please refresh p. 481-488.
And the first parts of Chapter 13.
Carbohydrates Start carbs at
238-248
Polysaccharides were discussed briefly (pp.
247-252).
the first
parts of Chapter 13.
Suggested
reading:GLYCOLYSIS Lehninger IV pp. 521-543 and pp. 560-571 and for regulation
of phosphorylase (pp
584-585).
In
terms of bioenergetics of glycolysis: sectiom on phosphoryl transfers
and ATP is relevant to glycolysis Lehninger
pp. 492-502.
=====================================================
READING SUGGESTIONS
Carbohydrates Start carbs at 238-248
Please refresh p. 481-488.
And the first parts of Chapter
13.
Suggested reading:GLYCOLYSIS
Lehninger IV pp. 521-543
In terms of bioenergetics of glycolysis:
sectiom on phosphoryl
transfers and ATP is relevant to glycolysis Lehninger pp. 492-502.
TCA cycle.
(reading 601-622)
Electron transport and Oxidative phosphorylation Lehninger IV
690-714
review
basic aspects of lipids Lehninger Chapt 10: pp. 343-350 and p. 355
FATTY ACID OXIDATION
=================================================================
BRIEF SYNOPSIS OF MATERIAL COVERED IN LECTURE
Oct 16 and 18
We reviewed basic
aspects of enzyme action – the active site and the forces that predominate in
the formation of the E.S. complex.
Reviewing the H-bond/electrostatic interaction/hydrophobic bond led to
the discussion of the effects of pH on enzyme activity.
We presented three
hypothetical examples showing the variation of activity as a function of pH. We discussed the variation of pK values for side chains with changing environment.
Temp. effects on enzyme activity with a mention of mesophiles and thermophiles.
Next we discussed
Inhibition – reversible and irreversible (and their distinguishing features)
Reversible inhibition: Kinetic pattern for competitive un-competitive, and mixed inhibitors. Discussed: Michaelis Menten and Lineweaver-Burke graphical methods.
Irreversible
inhibition: the example used was of a
catalytically essential cysteine side chain that was covalently modified by
iodoacetamide. We mentioned dialysis to
remove excess reagents and then how a series of inactivation curves could be
obtained using a series of iodoacetamide concs.
We mentioned the
expected dependence of the inactivation rate on the concentration of
iodoacetamide (reminding you of second order kinetics and pseudo first order
kinetics).
This led us to
affinity labels. The example quoted was
to attach a substrate-like fragment (-R) to the iodoacetamide –NH-R
functionality. Affinity labels typically
show saturation in their rate of inactivation vs. [Inhibitor] (rather like Michaelis Menten kinetics.
Mechanism-based inactivators were next.
We addressed why the term “suicide inactivator/inhibitor”
was inappropriate – the enzyme is a victim of deceit … the critical difference
between affinity labels is that mechanism-based inactivators
are activated as part of the enzyme’s catalytic mechanism, whereas affinity
labels already contain a reactive group before they arrive at the active site.
We discussed why
pharmaceutical companies generally prefer reversible over irreversible
inhibitors …
Proteases
The overall reaction
was described ... their critical importance in variety of cellular processes –
digestion, regulation, signalling, viral replication,
blood clotting .. etc.
Zymogens were covered together with the other
strategies that the cell takes to avoid autolysis. We mentioned chymotrypsinogen
and why it is inactive. The role of zymogen granules and the
presence of protease inhibitors.
A detailed
description of chymotrypsin mechanism followed:
First the enzyme
substrate encounter: how to calculate
how fast one would expect a diffusion controlled
reaction to be. What factors do the
various serine proteases exploit to achieve substrate discrimination/substrate specificity.
The catalytic triad
of a serine protease was discussed together with the evidence for it. The problems of abstracting a proton from a
serine hydroxyl using a histidine base were raised.
Next we will discuss
the covalent steps in chymotrypsin catalysis.
At the end of
Tuesday’s lecture (or Thursday at the latest) you should be able to handle
questions (some of them may be covered by Dr. B):
CHEM641 2nd exam Fall
05: 2a, 3, 4, 10, 11d, I, j, k,
CHEM527 Second Exam Fall 06: 1, 2 (a,b),
5, 12 (a, b, h),
<><><><><><>><><<<><><><><><><>>>
Dr. Bahnson has already
started Bioenergetics
Please refresh p. 481-488.
And the first parts
of Chapter 13.
<><><><><>>><<><><>>
CT will remind you
of a few key features …
After
bioenergetics/intro to metabolism we will start carbohydrates/glycolysis
Start carbs at 238-248
==================================================
October 23 2007
Continued
discussion of chymotrypsin mechanism – and the roles
of ASP, HIS and SER in catalysis of this serine protease. Mentioned
the tetrahedral intermediates and the acyl enzyme,
the numerous transition states etc.
Described the use of steady state and pre-steady state
methods in understanding enzyme catalysis. We recapped turnover number and showed that
one needs rapid reaction methods to obtain detailed information about enzymatic
catalysis. Discussed the burst-phase
kinetics with para-nitrophenylacetate and why esters
show burst phases and amides generally do not.
We discussed experiments with radiolabelled para-nitrophenylacetate which are consistent with a
covalent acyl-enzyme intermediate … and newer mass
spectrometry and crystallography techniques to show covalent
intermediates. We reviewed fluorophosphate inhibitors of serine proteases (introduced
substrate protection concept), nerve gases, TPCK and TLCK as affinity labels,
and a simple mechanism-based inactivator of chymotrypsin.
==================================================
October 25, 2007
We discussed other
classes of proteases (including other serine proteases trypsin,. Elastase and the evolutionarily
unrelated subtilisin)
A REMINDER – JUST IN
CASE IT WAS NOT COVERED EARLIER:
TRYPSIN hydrolyses
immediately C-terminal to ARG and
CHYMOTRYPSIN has a preference fro aromatic side chains and
hydrolyzes W-X-; Y-X and F-X (but not
when X=proline)
We mentioned convergent and divergent evolution again …
in context of serine proteases …
THIOL
PROTEASES: papain
and cathepsins
ASPARTYL PROTEASES:
we talked about pepsin, rennet/chymosin (in cheese
manufacture); renin in control of blood pressure; and
retroviral proteases
(in
latter context we discussed how one might design a protease inhibitor)
METALOPROTEASE: e.g carboxypeptidase and what it does
- - - - - - - -
PRE-METABOLISM/RECAP BIOENERGETICS
Reminder that free
energy depends on intrinsic chemical structure and concentration … / reminder
of le Chatelier’s principle … discussed the thermodynamic instability but
the kinetic stability of cellular biomolecules – and
a philosophical note on the struggle against equilibration ….
We described how one
can determine whether a reaction is at equilibrium or not in the cell …
freeze-clamping … and the significance of finding a reaction that lies very far
from equilibrium …
We mentioned
feedback inhibition and the control of reaction sequences by allosteric enzymes.
PRIMER ON CARBOHYDRATES
Roles
Trioses aldo
and keto …
Numbering of hexoses, chiral centers, glucose
and fructose …
Suggested reading:GLYCOLYSIS Lehninger IV
pp. 521-543
In terms of bioenergetics of glycolysis: sectiom on phosphoryl transfers and ATP is relevant to glycolysis Lehninger pp. 492-502.
==================================================================
October 30th and
Nov 1
Ring structures of hexoses, anomeric carbons. Glycosidic linkages.
Intro
to glycolysis.
Why would glycolysis
be used over the complete oxidation of glucose to carbon dioxide and
water. Glycolysis
can produce ATP very rapidly and this is advantageous under certain
circumstances.
The reactions of glycoysis were then discussed:
Hexokinase/glucokinase
Phosphoglucoisomerase
Phosphofructokinase
We discussed the
mechanisms of aldolases together with how the free
energy change of this reaction is influenced by the concentration of reactants
and products. We also discussed claims
that an active site cysteine was the catalytic base.
Triosephosphate isomerase
Glyceraldehyde 3-P dehydrogenase
and mechanism. The importance of thioesters.
Phosphoglycerate kinase
Phosphoglucomutase
Enolase
Pyruvate kinase
and the reasons why PEP has a high free energy of hydrolysis.
Lactate
dehydrogenase (and allusions to mechanism with reference to alcohol dH)
=======================================================================
November 5 and 8th
Thiamine
pyrophosphate as an essential cofactor (prosthetic group) of pyruvate carboxylase (thiamine is
the water soluble vitamin .. deficiency causes Beri-beri)
generating acetaldehyde from pyruvate. Acetaldehyde then serves as the electron
acceptor for NADH in yeast … generating ethanol.
Discussed three ways
to get rid of electrons generated during glycolysis
(return electron to pyruvate or acetaldehyde) or get
reduce oxygen to water using the electron transport chain (see later).
We discussed the
entry of other monosaccharides D-fructose, D-mannose,
D-galactose.
While mannose is converted to mannose 6P with hexokinase
the 2-OH can be easily epimerized because it is adjacent to the carbonyl oxygen
at C1. We discussed the more elaborate
way to epimerize the 4-OH of galactose (with the key
step being an internal oxidation-reduction using a bound molecule of NAD+.
The hydrolysis of
maltose, lactose and sucrose by glycosidases at the
brush-border was described. Lactose
intolerance was mentioned.
Polysaccharides were
discussed briefly (pp. 247-252).
Linear and branched,
homo- and heteropolysaccharides….
Amylose and amylopectin and glycogen.
We mentioned why
glucose is stored intracellularly as a polymer and discussed osmotic pressure
in general terms.
Finally we mentioned
cellulose – a linear polymer of beta 1-4 glycosidic
linkages … together with cellulases and
microorganisms that can degrade cellulose.
We discussed the
absence of cellulose degrading capabilities in humans and the consequences of
this …
The digestion of
dietary starch and glycogen was mentioned and distinguished from the
utilization of stored glycogen in a liver cell.
Reasons for the existence of phosphorylase
were proposed. The reaction mechanism
was discussed and the role of debranching enzyme and phosphoglucomutase were mentioned (see pp. 560-571)
Regulation of glycolysis at non-equilibrium steps was the next topic:
PFK and its complex
regulation involving allostery (we reintroduced T and
R states).
The hormonal and allosteric control of phoshorylase
was addressed (pp 584-585).
We then covered
tracing the flow of material through metabolic pathways using C isotopes
(stable and radioactive). Tracing of
examples in glycolysis was shown in class. Finally an example of the use of 13-C
isotopes in the diagnosis of ulcer type was mentioned (although this is not
directly relevant to glycolysis).
THIS CONCLUDES THE
MATERIAL FOR THE SECOND EXAM …
<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>
We next provided the handout – for the TCA cycle. (reading 601-622)
We discussed the pyruvate dehydrogenase multienzyme complex
spending quite a bit of time with
the figure at the bottom of page 605
We listed the cofactors of E1, E2,
and E3 and started discussing the deficiency diseases that accompany thiamine
and riboflavin. We discussed arsenic poisoning and the widely accepted mode of
action of arsenite.
We also talked about arsenic as a therapeutic (although this is not
directly related to the TCA cycle!).
We introduced general issues of TCA cycle, and then the enzyme steps in
turn. The prochirality of citrate was reviewed
together with: substrate level phosphorylation,
tracing radiolabels (common exam questions) and energy balance sheets. In
the latter we used 2.5 ATP per NADH generated in mitochondrion and etc (as in
text). In this manner the complete oxidation of a glucose molecule yields
30 ATP. We gave other examples of sample questions re energy yield.
The need for anaplerotic reactions was introduced and
one (biotin dependent pyruvate carboxylase)
was highlighted. Regulation of TCA cycle enzymes was mentioned. The
oxidation and energy yield of ethanol was given as an example of the oxidation
of alcohols. Methanol and ethyleneglycol
oxidation was mentioned and the metabolism of these poisons was addressed..
================================================================
Electron
transport and Oxidative phosphorylation Lehninger IV
690-714
We started with a discussion of how one might convert
pairs of electrons (i.e. NADH) into ATP and the thinking behind the chemical
coupling hypothesis and the difficulties which led to the proposal that ATP
generation is driven with a proton/electrochemical gradient. The anatomy
of the inner mitochondrial membrane was discussed with dissection into various
clusters of proteins (complexes I-IV). The main issues (mode of electron
transport in the electron transport chain; mechanism for extrusion of protons;
coupling between proton/electrochemical gradient and ATP formation) were
introduced and covered in turn.
Basic aspects of flavins, heme,
cytochromes, Fe/S centers, Quinones were
covered. The susceptibility of cytochrome
oxidase to inhibition was mentioned. A simplified view of proton export
coupled to electron transport utilized complexes I and III
communicating via coenzyme Q.
ATP synthase was introduced with emphasis on
how one might generate ATP using perturbation of the equilibrium ADP + Pi = ATP
+ H2O by preferential binding of ATP. And
how conformational change driven by proton transport might allow release of ATP
before rehydrolysis.
Inhibitors of oxidative phosphorylation were
mentioned including cyanide, CO, H2S. Uncouplers:
synthetic – like the nitrophenols
=============================================================================================================
Nov 26 EXAM handed back …
>>KEY to second exam KEY_2ndexamCHEM641_07F.pdf
Please consult key before
raising grading issues
As always, we will promptly
address grading errors.
Please note,
there was a significant typo. in ONE part of a question that was noticed by one of you (thanks!). I did not announce my mistake because about
20 people had already handed in their exams before the error was apparent. I have ignored this part of the question in
grading. Sorry for the anguish.
Average 68.5% (higher than usual
– as someone observed “the exam was predictable”)
LOW 26%
HIGH 99%
===============================================================================================================
READING FOR FATTY ACID OXIDATION … we first need to review basic
aspects of lipids Lehninger
Chapt 10: pp.
343-350 and p. 355
This will introduced:
neutral
fats (triacylglycerols – and their constituent fatty
acids)
phospholipids
waxes
steroids
============================================================================================================
Next … FATTY ACID OXIDATION READING: Lehninger IV
pp. 631-639; 643, 650-652
We discussed sources of fatty acids – dietary and
storage … we mentioned, but did not discuss further, fatty acid biosynthesis
We dealt with the digestion of dietary neutral fat .. the role of natural
detergents, the initial lipase attack on triacylglycerols,
lipase action on chylomicrons.
We mentioned phospholipases
too (belatedly).
Carnitine shuttle and the 4 separate activities that comprise
fatty acid oxidation (beta-oxidation). Acyl-CoA dehydrogenase, enoyl-CoA hydratase, hydroxy-acyl-CoA
dehydrogenase and thiolase.
The first three (E1, 2, 3) have analogies to succinate
dH, fumarase
and malate dH respectively.
We showed the mechanism of each one …
We discussed ATP yields from a C-16 CoA and etc ….
A fairly common genetic disease MCAD deficiency was
described (medium chain acyl-CoA dH deficiency).
The symptoms and the treatment options. We mentioned that about 2% of SIDS cases are
likely to be MCAD related.
Finally we spent a while talking about Ackee fruit, the poison therefrom
(hypoglycin) and the toxic metabolite it is converted
into MCPA-CoA.
MCPA-CoA was shown to be a mechanism-based
inhibitor of the acyl-CoA dehydrogenase – we
explained why. The symptoms of Jamaican
vomiting sickness were explained.
=========================================================================================================
Tuesday: we then discussed the biosynthesis of ketone bodies from excess acetyl-CoA
... and how acetoacetate, hydroxybutyrate
and acetone were formed. We mentioned
fasting and starvation and the diabetic condition.
Amino
acid catabolism: Finally we very briefly outlined the pathways
for the catabolism of amino acids. First
we addressed the issues of disposing of the N- (initially via transaminases and glutamate dehydrogenase – and then via
the urea cycle or uric acid generation or via release of free ammonia in some
aquatic animals).
Then we mentioned the fate of the carbon skeleton (you
can see this on page 671). Here we gave
three examples. The first two were
straightforward (alanine and aspartic acid).
In contrast, isoleucine metabolism
require multiple steps and eventually yields acetyl-CoA
and succinyl-CoA (we know how these are
metabolized!). Finally we mentioned the
court case in which a woman was accused of poisoning her baby with ethylene
glycol when, in
reality the child had a deficiency of the last enzyme of the ILE breakdown
pathway. This was featured on page 684 of the
book.
We are not going to test on the details of amino acid
oxidation and the urea cycle (please wait for CHEM643).
FINALLY REMEMBER THAT THE EXAM MATERIAL IS INSPIRED BY
WHAT HAS BEEN COVERED IN LECTURE … OR QUESTIONS INSPIRED THEREFROM … OR
QUESTIONS ON PREVIOUS TESTS.
<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>
Good luck!
CT
.