Transcript Carbohydrate Metabolism

Clinical Biochemistry 6th semester Spring 2016
Week
Theoretical
Lecturer
Tutorial
Practical
1 [28 Jan]
CHO metabolism
Prof. Gad
---
----
2 [31 Jan- 4 Feb]
CHO metabolism
Prof. Gad/Dr. Ingy
Glycolysis
•Creatinine and urea assay
3 [7-11 Feb]
CHO metabolism
Prof. Gad/Dr. Ingy
Aerobic
metabolic
and
explanation
of
student
presentations
Urine analysis
4 [14-18 Feb]
CHO metabolism
Prof. Gad/Dr. Ingy
glycogen & gluconeogenesis
•Fragility test
•Effect of different of haemolytic agents
•Determination of RBCs count
• First quiz: Will be informed
5 [21-25 Feb]
Disorders of CHO metab.
Prof. Gad/Dr. Ingy
HMP shunt and Cells adapted
to function
Hb assay, ESR & hematocrit
6 [28 Feb- 3 Mar]
Disorders of CHO metab.
Prof. Gad/Dr . Ingy
Clinical cases on diabetes
Oral glucose tolerance test
Midterm exams [5-14 Mar]
7 [12-17 Mar]
Oxidative phosphorylation
(Compensation)
Prof. Hans
---------
Glycated Hb
8 [20-24 Mar]
Lipid metabolism
Dr. Sahar
Q & A on oxid. phosphorylation
Extraction of PC from egg yolk
Extraction of cholesterol from egg yolk
Performing solubility tests on different lipids
(compensation on Sat 26 Mar)
9 [27-31 Mar]
(27 Mar off)
Lipid metabolism
(compensation)
Dr. Sahar
Student Presentation
(compensation on Sat 26 Mar)
Determination of TG, Determination of
cholesterol and HDL –C (Combined lab)
10 [3-7 Apr]
Lipid metabolism
Dr. Sahar
Student Presentation
• Revision
Dr. Sahar
metabolism
Dr. Lipid
Mohamed
Z Gad
• Second quiz: Will be informed
11 [10-14 Apr]
Disorders of
6 April 2017
12 [17-21Apr]
lipid metab.
Integration of metabolism
6 April 2017
Prof. Hans
Clinical cases on lipid disorders
Dr. Mohamed Z Gad
 Practical exam days
 Practical exam days
1
1
Email: [email protected]
Day off: Mostly Thursday
6 April 2017
Dr. Mohamed Z Gad
2
A Map of The Major Metabolic
Pathways in A Typical Cell
6 April 2017was taken from “Biochemistry”
Dr. Mohamed
Z Gad
The Diagram
by Voet
& Voet (2nd edition, 1995, John Wiley 3&
Sons, pg. 413)
Lecture 1 PHBC621
Carbohydrate
Metabolism
(Glycolysis)
6 April 2017
Dr. Mohamed Z Gad
4
Learning Objectives
By the end of this session you should be able to:
• Outline the three stages of glycolysis.
• Describe the steps of glycolysis between glucose and
pyruvate and recognize all the intermediates and
enzymes and the cofactors that participate in the
reactions.
• Mention ATP-generating reactions.
• Illustrate the regulation of glycolysis.
6 April 2017
Dr. Mohamed Z Gad
5
Major Pathways of CHO Metabolism
CHO metabolism in mammalian cells can be Glucose is the major fuel of most
organisms. The major pathways of CHO
classified into:
1. Glycolysis: Oxidation of glucose to metabolism either begin or end with
glucose.
pyruvate (aerobic state) or lactate
(anaerobic state)
2. Krebs cycle: After oxidation of pyruvate
to acetyl CoA, acetyl CoA enters the Krebs
cycle for the aim of production of ATP.
3. Hexose monophosphate shunt: Enables
cells to produce ribose-5-phosphate and
NADPH.
4. Glycogenesis: Synthesis of glycogen from
glucose, when glucose levels are high
5. Glycogenolysis: Degradation of glycogen
to glucose when glucose in short supply.
6. Gluconeogenesis: Formation of glucose
from
noncarbohydrate sources. Dr. Mohamed Z Gad
6 April 2017
6
Glycolysis (Embden-Meyerhof Pathway)
[glycolysis: from the Greek glyk-, sweet, and lysis, splitting]
Glycolysis occurs in all human cells. Glycolysis is believed to be
among the oldest of all the biochemical pathways.
Aerobic: Glucose  Pyruvate
Anaerobic: Glucose  Lactate (or ethanol & acetic
acid)
Glycolysis (10 reactions in 3 stages, all in cytoplasm)
1) Priming stage: D-Glucose + 2ATP  D-fructose 1,6-biphosphate + 2ADP + 2H+
2) Splitting stage : D-Fructose 1,6-biphosphate  2 D-Glyceraldehyde 3-phosphate
3) Oxidoreduction – Phosphorylation stage:
2 D-Glyceraldehyde 3-phosphate + 4ADP + 2Pi + 2H+  2Lactate + 4ATP
----------------------------------------------------------------------------Sum:
Glucose + 2ADP + 2Pi ----- 2 Lactate + 2ATP + 2H2O (Anaerobic)
+ ---- 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H O
Glucose
+
2ADP
+
2Pi
+
2NAD
2 7
6 April 2017
Dr. Mohamed Z Gad
(Aerobic)
Common Abbreviations & Alternative Names
6 April 2017
Dr. Mohamed Z Gad
8
1) Priming Stage
• Glucose (and other hexoses) are
phosphorylated immediately upon
entry into the cell. Phosphorylation
prevents transport of glucose out of
the cell and increases the reactivity
of oxygen in the resulting phosphate
ester.
• Several isoenzymes of hexokinase
with different Km values for glucose
are located in different tissues. Brain
hexokinase has a particularly low
Km for glucose.
• The
major
enzyme
for
phosphorylating glucose in liver is
glucokinase.
• Steps
by hexokinaseDr.&
6 April catalyzed
2017
Mohamed Z Gad
PFK-1 are irreversible.
9
Differences between Glucokinase & Hexokinase
Hexokinase
Glucokinase
 Present in all tissues
 Liver only
 Low Km for glucose (<0.1  Higher Km for glucose
mM)
 Strongly inhibited by G6P
 Not inhibited by G6P
 Non-inducible enzyme, not  Inducible, synthesis induced
affected by diabetes or
insulin & repressed in diabetes
insulin
 Level of enzyme is not  Depends on glucose concentration
affected by fasting or high
CHO diet
 Act on glucose, fructose and  Glucose only
6 April 2017
Dr. Mohamed Z Gad
galactose
10
by
2) Splitting Stage
• The reaction catalysed by aldolase is
the reverse of aldol condensation.
• Although the cleavage of F1,6BP is
energetically
unfavourable,
rapid
removal of the product drives the
reaction forward.
• Of the two products of the aldolase
reaction, only GAP (or G3P) serves as a
substrate
for the next reaction in
glycolysis.
• To prevent the loss of the other threecarbon unit, triose phosphate isomerase
catalyses the interconversion of DHAP
& G3P. Because of this reaction, the
original molecule of glucose has now
been
converted to two molecules
of
6 April 2017
Dr. Mohamed Z Gad
G3P.
11
3) Oxidoreduction –
Phosphorylation Stage
• G-3-P dehydrogenase is a tetramer,
each subunit contains 1 binding site
for G3P & another for NAD+ (NAD+
is permanently bound to the enzyme).
• G3P  1,3-BPG  3PG and PEP 
Pyruvate are examples of “substrate–
level” phosphorylation.
• 3-PG  2-PG is mediated by an
intermediate [2,3-BPG]. Most cells
have low amounts of 2,3-BPG except
in RBCs, which act as allosteric
modifier of Hb-O2 binding.
• PEP  Pyruvate is an “irreversible
reaction” due to free energy loss
associated
theMohamed Z Gad
6 April 2017with tautomerization of Dr.
enol to the more stable keto form.
12
Study Question ?????
What is meant by “substrate–
level phosphorylation” ?
ADP is converted to ATP by the direct
transfer of a phosphoryl group from a high
energy compound.
6 April 2017
Dr. Mohamed Z Gad
13
Study Question ?????
Why
glycolysis
under
anaerobic conditions proceed
to lactate and not just stop at
pyruvate formation ?
The reaction of lactate dehydrogenase is essential in
anaerobic glycolysis , as it is the mean for reoxidizing
NADH formed in the G-3-P dehydrogenase step to reenter into the glycolysis cycle. In aerobic glycolysis
reoxidation takes place in mitochondria by the respiratory
chain.
6 April 2017
Dr. Mohamed Z Gad
14
Overall pathway of Glycolysis & ATP
Formation
Number of ATP generated from
glycolysis
Enzyme
Aerobic
Anaerobic
Hexokinase
-1 ATP
-1 ATP
PFK-1
-1 ATP
-1 ATP
G-3-P
dehydrog.
+6 ATP
----
Phosphoglycerate
kinase
+2 ATP
+2 ATP
Pyruvate
kinase
+2 ATP
+2 ATP
Sum 6 April 2017
+8 ATP
+2 ATP
Dr. Mohamed Z Gad
15
Study Question ?????
Louis Pasteur, the great 19th century French
chemist and microbiologist, was the first scientist
to observe the following phenomenon. “Cells that
can oxidize glucose completely to CO2 and H2O
utilize glucose more rapidly in the absence of O2
than in its presence”. It would appear that O2
inhibits glucose consumption. Thus, another
definition for Pasteur effect is: inhibition of
glucose utilization and lactate accumulation by the
initiation of respiration (O2 consumption)… Can
you explain why ?
Louis Pasteur
(1822 - 1895)
The reason behind this phenomenon is that complete oxidation of glucose under
aerobic conditions yield much more ATP (~38 ATP) than anaerobic glycolysis
(~2ATP). Thus it is anticipated that the rate of glucose consumption will be 1920 times faster under anaerobic condition to meet the metabolic demand in a
6 April 2017
Dr. Mohamed Z Gad
16
way equivalent to aerobic conditions.
Regulation of
Glycolysis
Rate of glycolysis is controlled primarily by
allosteric regulation of the 3 key enzymes
(irreversible steps), hexokinase, PFK-1, and
pyruvate kinase.
• PFK-1 is the major regulatory Enzyme
Activator
enzyme of glycolysis. In the liver
Hexokinase AMP, ADP, Pi
only, PFK-1 is activated by fructose2,6-diphosphate (F-2,6-DP).
PFK-1
F-6-P, AMP,
F-2,6-DP
• PFK-2, the enzyme that synthesize
(liver only)
the activator F-2,6-DP, is itself a
AMP, F-1,6regulatory enzyme. It is inhibited by Pyruvate
DP
citrate
&
ATP
and
by kinase
phosphorylation. The reverse reaction
is catalyzed by fructose-2,6diphosphatase(F-2,6-DPase).
• Hormones also regulate glycolysis
e.g., glucagon inhibits glycolysis by
repressing the synthesis of F-2,6-DP.
Insulin promotes glycolysis by
stimulating
6 April 2017 the synthesis of F-2,6-DP.
Dr. Mohamed Z Gad
Inhibitor
G-6-P
NADH,
H+,
citrate, ATP
ATP,
acetyl
CoA,
phosphorylation
17
Study Question ?????
What effects do fluoride
magnesium have on glycolysis ?
6 April 2017
Dr. Mohamed Z Gad
and
18
Comments on Glycolysis
 Glycolysis is the only pathway that produce ATP in absence of O2.
 The best known inhibitors of the glycolytic pathway include:
2-Deoxyglucose: causes inhibition of hexokinase.
Sulfhydryl reagents (e.g. Hg-compounds and alkylating agents as
iodoacetate); inhibit glyceraldehydes-3-phosphate dehydrogenase
which has cysteine residue in the active site.
Fluoride: a potent inhibitor of enolase. Thus, fluoride is usually added
to blood samples to inhibit glycolysis before estimation of blood
glucose.
 Magnesium: required for kinase reactions by forming Mg-ATP
complex.
 Accumulation of lactate is responsible for muscle fatigue and cramps
•
observed
under heavy exercise (anaerobic glycolysis).
 In6 April
RBCs,
glycolysis is theDr.major
of ATP since RBCs 19lack
2017
Mohamed Zsource
Gad
mitochondrial oxidation.
Animation of Glycolysis
glycolysis 5.swf
6 April 2017
Dr. Mohamed Z Gad
20
‫كلمات باقية‬
‫للعظيم قلبان‪ :‬قلب يتألم … و … قلب يتأمل‪.‬‬
‫جبران خليل جبران‬
References for Further Readings:
Latest editions of:
•Biochemistry by L. Stryer, Freeman & Company New York ….
•Harper’s Biochemistry by R.K. Murray, D.K. Granner, P.A. Mayes &
V.W. Rodwell. Biochemistry. Appleton & Lange, New York, Connecticut,
California.
•Biochemistry by T. Mckee & J. Mckee, Wm. C. Brown Publishers,
London, Madrid, Tokyo ….
•Biochemistry by I.D.K. Halkerston, Second Edition, John Wiley & Sons
Inc., Williams E Wilkins, USA.
• Textbook of Biochemistry With clinical Correlations by T.M. Devlin,
Wiley-Liss Publication, New York, Toronto ….
•Biochemistry Illustrated by P.N. Campbell & A.D. Smith, Longman
Group UK Ltd, UK.
•Principles
Of Biochemistry byDr.G.L.
Zubay, W.W. Parson & D.E. Vance,
6 April 2017
Mohamed Z Gad
22
Wm. C. Brown Publishers, Iowa, Melbourne, Ox for d.
6 April 2017
Dr. Mohamed Z Gad
23