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Bahnson Links KEY to SECOND EXAM CHEM 641 Second Exam F05.pdf ANSWER KEYS TO SIX OLD
EXAMS Partial answer key: 15th Class - We will start the Thorpe
part of CHEM641 continuing (more or less) where Dr. Bahnson
left off – there will inevitably be some overlap at the beginning. CT cannot cover all of
the Text … if we are going to get to fatty acid oxidation before it
starts snowing. Where possible, I will
refer to pages from the Text that I especially recommend that you
peruse. I will try and identify
questions from the book that are appropriate to help with the material
– although the style of questions from the text and those found in exam
2 are likely to be different. The sort
of questions I ask may be found on the posted exams on the Web site - I will also try to identify pertinent
questions from these exams where appropriate.
These should substitute for practice problems. Answers will be given later – but please
try the questions first! More or less in order
of appearance in the remainder of the Enzymes section: Review of typical active site side chains and
predicting the pH effects on enzyme activity (p. 420). Exam Questions 527/04, Final; Q6 527/03, Final Q10 641/98, (2nd ex) Q5 641/94, (2nd ex) Q2 641/96, (Final) Q10 641/94, (Final) Q17 (You need to be
comfortable with pH and pK and ionic forms of amino
acid side chains.) Temperature effects on catalysis p.
420/421 Enzyme Inhibition: 421-426. We essentially finished
enzyme "reversible" inhibition on Tuesday. 16th Class -
Dr. Bahnson
has already talked about the catalytic strategies employed by an alcohol
dehydrogenase. We will discuss in some
detail one other class of enzymes here – the proteases with special
emphasis on the serine proteases. Read
p. 453-462. What we covered: Irreversible inhibition
with a group-specific reagents like iodoacetamide
or iodoacetic acid (structure p. 90). The expected kinetics for such an inhibitor (overall second-order,
but pseudo-first order if the inhibitor is in considerable
excess). The general lack of
specificity of these sorts of compounds.
The increased specificity expected for affinity labels –
their design and kinetic behavior).
Then mechanism-based inactivators
(real examples of appropriate inhibitors will be presented later). Why the pharmaceutical industry generally
prefers non-covalent inhibitors. Next,
we discussed general aspects of proteases: including physiological roles, zymogens
and protein secretion. We started
talking about the mechanism of a serine protease – chymotrypsin. First refreshing concepts of diffusion
with respect to binding substrates to active sites. The concept of a physical limitation for
the upper limit of catalytic proficiency. We mentioned the ways proteases can
discriminate between various substrates and started our discussion of the catalytic
triad. I will identify problems
when we have finished chymotrypsin. 17th Class - We dealt with the following topics today. The chemical mechanism of a typical serine protease, chymotrypsin. Including the two tetrahedral intermediates, the acyl enzyme and the roles of the catalytic triad in this multistep reaction. We discussed the difference between steady-state and pre-steady state kinetics and how delving deeply into enzyme mechanisms needs the latter, as well as the former. We mentioned the use of substrate analogs (fluorescent and the p-nitrophenyl-esters) as probes of mechanism. A description of the burst phase with p-nitrophenylacetate was followed by discussion of what constitutes evidence for a covalent enzyme intermediate (examples of experiments were given). Fluorophosphate derivatives were discussed as covalent inhibitors of serine proteases (and of other hydrolases that may use a particularly reactive serine in the context of a catalytic triad). Acetylcholinesterase and nerve agents were discussed. We then discussed the affinity labels TPCK and TLCK directed towards chymotrypsin and trypsin respectively. Some questions from old exams (Answers are at top of this page): 641/98,
(2nd ex) Q2, 4c 641/94,
(2nd ex) Q5c,d, Q6 641/98,
Q3, 641/94,
(2nd ex) Q1 641/94
(Final) Q8 18th Class -
the various classes of serine proteases
(including the evolutionary-convergent subtilisin);
the thiol proteases (including papain and
various cathepsins); the aspartyl
proteases (like pepsin, rennet, rennin and certain viral proteases); and
the metalloproteases (exemplified by carboxypeptidase).
General comments about these diverse strategies for hydrolyzing
peptide (and ester) bonds followed. <<<<<<<<<<<<<<<<<<<<<<<<<
>>>>>>>>>>>>>>>>>>>>>>>>>> Next we
reviewed concepts of bioenergetics – how free energy changes
depend on intrinsic structure and concentration. The important difference between
thermodynamics and kinetics was mentioned for the umpteenth time. We then started a discussion about how one
experimentally measures whether reactions in metabolic pathways are really at
equilibrium. The concept of mass action
ratio will be amplified next lecture. <<<<<<<<<<<<<<<<<<<<<<<<<
>>>>>>>>>>>>>>>>>>>>>>>>>> Some suggested questions for the enzymes
section of Thorpe's part (including some ones already mentioned): 641/98, (2nd ex)
Questions: 2; 3; 4a-c; 5a-c; 7; 641/94, (2nd ex)
Questions: 1; 2, 5b, 5c, 5d 5c,d, Q6 641/94 (Final) Q8, 9, 17
a, b, e 641/96 (Final) Q 10 b, e, g; Q 10 a, d, e 527/03 (Final) Q 9 d, g; 10 527/04 (Final) Q 6, 8 d <<<<<<<<<<<<<<<<<<<<<<<<<
>>>>>>>>>>>>>>>>>>>>>>>>>> 19th Class - Tues. 11/1/05 Carbohydrates were reintroduced as
a prelude to considering the chemical steps of glycolysis. The following topics (suggested book
section 203-210) were covered aldo- vs keto sugars, trioses; the ring structure of common aldohexoses
and ketohexoses; hemiacetal and hemiketals;
pyranose and furanose;
numbering carbons; anomers; brief intro to glycosidic bonds.
(We defer di- to polysaccharides until after
core reactions of glycolysis have been covered). Introduction to glycolysis and the fate of pyruvate was given. Critical issue is not only the availability
of oxygen but the flux of ATP needed per second. The trade-off between efficiency
(ATP/glucose) and ATP production rate (ATP/sec) was emphasized with examples.
21st Class - Tues. 11/8/05 22nd Class - Thurs. 11/10/05 The rationale for storing glucose in polymer form was presented together with a description of phosphorylase and debranching enzyme catalysis. We dealt with cellulose and cellulases briefly. Control of glycolysis was covered with special
emphasis on PFK and its allosteric regulation by
adenine nucleotides and Fructose 2,6-diP. The various modes of regulation of phosphorylase were covered – and this should
complement Dr. Bahnson's sections on this
enzyme. Finally the use of isotopes to
help trace biochemical pathways was presented. We also discussed the use of isotopes in
medical diagnosis.
A note
about the second exam The exam is out of 100 points. The section of enzymes and the section on glycolysis/carbohydrates carry about equal weight. There are miscellaneous minor bits and
pieces making up the balance. Coverage:
classes 15-22 inclusive. Reading: (I
put pages on blackboard – but here they are again – in the order
that we covered the topic). Pages:
420-426 // 453-462 // 203-210 // 215-233 // 538-552 // 555-559 //
578-604 Material
covered after coverage of second exam TCA
cycle: Pages 608-634 Remember metabolic charts provided on
exam. We discussed the logic of the
pyruvate dH multienzyme
complex with its multiple coenzymes/cofactors/prosthetic groups. Riboflavin, niacin deficiency, arsenic
poisoning 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. Chapter 20 Oxidative Phosphorylation (pages 640-642 and 645-to end of chapter) 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 and FCCP, and natural – like thermogenin in (e.g.) brown fat. Oligomycin inhibits ATP synthase. Fatty Acid Oxidation We started with a brush-up of lipids (Chapter 8 to page 254). Neutral fat, structures and properties of fatty acids, phospholipids, waxes and steroids. Reminded of difference between saponifiable and non-saponifiable lipids. Beta-oxidation reading Chapter 23: 738-751 and 759-761 Lipases and the absorbtion of neutral fats. Transport in blood stream. Adipose tissue and the hormonally controlled lipases therein. Beta oxidation proper with only a brief mention of the carnitine shuttle system. Refer to the metabolic chart for beta oxidation. Discussed the acyl-CoA synthetases and the hydrolysis of pyrophosphate. The reactions of E1-4 drew curved arrows for E1 and E4. Discussed energy yields of palmitoyl-CoA (C16) 108 ATPs; hexanoyl-CoA (C6) 38. Discussed acyl-CoA dehydrogenase deficiencies (medium chain acyl-CoA dehydrogenase deficiency) and the likely consequences of an impaired fatty acid oxidation pathway. A natural inhibitor of fatty acid oxidation – the poison hypoglycin from the Ackee – showed that this is a mechanism based inactivator of E1. Predicted symptoms and treatment. Ketone bodies were discussed – and their interrelation with anaplerotic reactions.
Fasting and diabetes
The Thorpe part of the Final exam will be worth 75 points. About 50 points will be on new material, and the remainder from topics covered in the second exam. Unlike previous years, there will be no "joint" part of the exam (no Bahnson/Thorpe hybrid questions). So
Dr. Bahnson's part will be 75 points too and
independent in coverage. Click
Here to go to archive of
Bahnson Links
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