Q: About Michaellis Constant (Km)


Enzyme Question No.10 (E-10)

 

 

The measuring of the Km in an isomerase that catalyzes the transformation of different D-carbohydrates in the corresponding L- isomers,  show different values, depending on the carbohydrate that is transformed. Given the following carbohydrates  with the corresponding values of Km, mark the one for which the enzyme show less affinity:

 

a) D-glucose                    Km =  2000 uM

 

b) D-galactose                Km =   4500 uM

 

c) D-mannose                Km =  8000 uM

 

d) D-Ribose                    Km = 10000 uM

 

e) D-fructose                   Km =  9000 uM

 

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Energetic Balance of the total oxidation of one mol of glucose up to CO2 and H2O: Understanding the contradictions.


 

For understanding the process of total oxidation of glucose, it is necessary to consider the different steps and metabolic pathways involved as well as the cellular location of these processes:

 

   

STEPS

M. PATHWAY

CELLULAR LOCATION

Glucose to  Pyruvate

Aerobic Glycolysis

Cytosol

Pyruvate to Acetyl CoA

Oxidative Decarboxylation of Pyruvate

Mitochondria

Acetyl Co A to CO2

Krebs Cycle

Mitochondria

  

 

Aerobic Glycolysis describes the oxidation of one mol of Glucose (6 carbons)  up to the formation of two moles of pyruvate (3 carbons each).

This conversion involves different reactions where ATP is produced or consumed:

Glucose to Glucose-6-(P)                                                                          – 1 ATP

 

Fructose-6-(P) to Fructose 1,6- bisphosphate                                 – 1 ATP

 

Since Fructose 1,6 bisphosphate becomes two trioses phosphate, the reactions after the aldolase reaction occurs twice (one for each triose that continues the glycolysis). In one reaction, NADH.H+  is produced; in other two reactions, a Substrate Level Phosphorylation (SLP) occur

 (2) Gliceraldehide 3 (P) to (2) 1,3 bisphosphoglycerate           +(2) NADH.H+

 

(2) 1,3 bisphosphoglycerate to (2) 3 phosphoglycerate (SLP*)     + 2 ATP

 

(2) Phosphoenol pyruvate to Pyruvate (SLP*)                                    + 2 ATP

 

(*Maybe it is necessary to recall now that there are two different ways of synthesizing ATP:  (a)SLP, in which ATP is synthesized using energy from some reactions of metabolism, like these reactions, and (b) Oxidative Phosphorylation, synthesis of ATP using the energy released in the Respiratory chain, from the oxidation of reduced cofactors)

 

Total energetic Balance from Glucose to (2) Pyruvate

(Aerobic Glycolysis): 

   _ 2 ATP  +  2 NADH.H+  + 4 ATP  = 2 ATP + 2 NADH.H+ in cytosol

 

 

An excellent animation of this process up to pyruvate can be found here.

 

Pyruvate will enter the mitochondria and will experiment Oxidative decarboxylation, in a reaction catalyzed by the pyruvate dehydrogenase complex.

The global reaction is:

2 Pyruvate + 2NAD+  +2 CoA —à 2 Acetyl CoA + 2 CO2 + 2 NADH.H+

This reaction occurs twice since each glucose (6 carbons) produce 2 pyruvates (3 carbons each), consequently these process produce

2 NADH.H+ in the mitochondria

 

Each Acetyl Co A is oxidized  in the Krebs Cycle yielding:

 

2 Isocitrate to 2 alfaketoglutarate            (+2CO2)                     2 NADH.H+

2 alfaketoglutarate to 2 Succinyl CoA (+2CO2)                          2 NADH.H+

2 Succinyl CoA to 2 Succinate (SLP)                                               2 GTP

2 Succinate to 2 Fumarate                                                                 2 FADH2

2 malate to 2 oxalacetate                                                                   2 NADH.H+

 

An animation with these reactions can be found in this link. 

Observe that at the end of the Kreb Cycle the 6 carbons of glucose have been oxidized to 6 CO2

 

At the same time, from the energetic point of view:

4 ATP have been obtained through Substrate level Phosphorylation (ATP synthesis without intervention of the energy of respiratory chain): 2 were obtained during the Aerobic Glycolysis and 2 were obtained in the Krebs Cycle as GTP (1 GTP is equivalent, from the energetic point of view,  to 1 ATP).

2 FADH2 have been obtained from the Krebs Cycle

8 NADH.H+ have been obtained inside the mitochondria (2 from Oxidative decarboxylation of Pyruvate and 6 from the Krebs cycle)

2 NADH.H+have been obtained in the cytosol through the Aerobic Glycolysis.

 

It is important to do these distinctions about the cellular location of the NADH.H+. since those produced in the cytosol should enter the mitochondria to be oxidized. Since the internal mitochondria membrane is impermeable to these nucleotides, (the mitochondria has their own pool of NAD) the NADH.H+ produced in the cytosol should enter using one of the shuttles already described for transporting the reduction equivalents of cytosolic NADH.H+  through the internal membrane of the mitochondria:

 

a)     the malate aspartate shuttle.

b)     The glycerophosphate shuttle

 

As described in other post, the malate-aspartate shuttle regenerates NADH.H+  inside the mitochondria, the energy yielding of the cytoplasmatic NADH.H+  is the same as if it was generated directly in the mitochondria

 

With the glycerophosphate shuttle, the reduction equivalents of the cytosolic NADH.H+  are transferred to FAD in the inner membrane. It means that the cofactor that will be oxidized in the respiratory chain when this shuttle is used, is FADH2 (see the explanation in this related post)

 

In conclusion, when the malate aspartate shuttle is used for transporting the reduction equivalents from the cytosolic NAD+ inside the mitochondria, we can consider that the t oxidation of glucose has produced:

4 ATP

10 NADH.H+  to be oxidized in the Respiratory chain

2 FADH2 to be oxidized in the Respiratory Chain.

 

If we use the convention that each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP, the total ATP production is:

 

Substrate Level Phosphorylation (SLP)                           04 ATP

10 NADH.H+  x 3 ATP/NADH.H                                          30 ATP

02 FADH2  x  2 ATP/FADH2                                                04 ATP

 Total                                                                                           38 ATP

 

Using the same convention (each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP,), but now assuming that the shuttle use is the glycerophosphate shuttle:

 

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 3 ATP/NADH.H                                        24 ATP

04* FADH2  x  2 ATP/FADH2                                            04 ATP

 Total                                                                                         36 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 36-38 moles of ATP (since it depends on the shuttle that is used for entering the reduction equivalents of the NADH.H+ produced in the cytosol through glycolysis).

 

Other possible results:

 

As described in other post, some books use the convention that each mol of NADH.H+, when oxidized in the respiratory chain, produce approximately 2.5 moles of ATP, while each mol of FADH2 produce 1.5 moles of ATP. Using this convention:

When the malate-aspartate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

10 NADH.H+  x 2.5 ATP/NADH.H                                     25 ATP

02 FADH2  x  1.5 ATP/FADH2                                           03 ATP

 Total                                                                                         32 ATP

 

When the glycerophosphate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 2.5 ATP/NADH.H                                    20 ATP

04* FADH2  x  1.5 ATP/FADH2                                        06 ATP

 Total                                                                                        30 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 30-32 moles of ATP

 

An advice: When solving a problem of this kind is absolutely necessary to know the conventions used for the yielding of the reduced cofactors (2.5 or 3 ATP/ NADH.H+ ? 1.5 or 2 ATP/ FADH2?) and the kind of shuttle that has been used for entering the reduction equivalents from the cytosol to the mitochondria.  A fair question will have both information. If not information is provided in the question, use the conventions followed by your professor during the lectures.

For students preparing for USMLE exams, the most used review books, like Harvey and Champe, in the “Lippincott Illustrated Reviews” of Biochemistry, Dawn B. Marks in “Biochemistry, Board Review Series”, Kaplan Biochemistry Lecture Notes for USMLE and “First Aid for the USMLE Step I”, agree in using the equivalence of  ‘approximately” 3 ATP for each NADH.H+  that is oxidized and ‘approximately” 2 ATP per FADH2. 

 

Related post:

To understand the mechanism of the shuttles and their difference in yielding ATP, I strongly recommend to read this post.

Metabolism of chylomicrons


 

Once dietetic lipids have been hydrolyzed and absorbed, the fatty acids are reesterified to other lipid products in the intestinal cells, to form new neutral fats, new cholesterol esters, new phospholipids and other lipids.

 

These new molecules, formed inside the intestinal cell, should be transported to different tissues through the polar environment of blood, so it is necessary to assamble, inside the intestinal cells,  these lipidic molecules with amphipatic apolipoproteins, to form Chylomicrons.

  

These chylomicrons, very rich in triacylglycerols, already contain apoproteins A  and apoB-48, an apoprotein that is exclusive of chylomicrons (Apo B-48 belongs to the same family that Apo B-100, but does not show the domain that is necessary to bind to the B-100 receptor). These chylomicrons do not contain yet Apo C-II, and they are called “nascent chylomicrons” 

 

Nascent chylomicrons are secreted by the intestinal cells and are transported via the limphatic system to the blood, where they will pick Apoprotein C-II and Apo E.  

Apolipoproteina C-II, as part of chylomicrons, activates Lipoprotein Lipase, an enzyme attached to the lumenal surface of the endothelial cells of capillaries. This enzyme catalyzes the hydrolysis of the triacylglycerols in the chylomicrons. 

 

 

 

Released fatty acids and glycerol from the hydrolized acylglycerols are uptaked by adipose cells, muscle and other tissues near the capillaries. Glycerol is also used heavily by the liver and kidneys and can continue glycolysis. Hydrolysis of chylomicron’s neutral fat provokes also the release of Apo A and Apo C-II and the shrink in size of chylomicrons, that become now “chylomicron remnant”, with lipids core that have a high proportion of cholesterol.

 

Chylomicron remnants  will be taken up by liver cells.

   

The hepatic uptake of chylomicrons remnants is based on a receptor-mediated endocytosis, that involves the recognition of chylomicron remnant ApoE by receptors on the surface of liver cells. It  should be noted that throughout this process, chylomicrons have acted as a means of transportating the component of lipids in diet, from the intestinal cells to peripheral tissues as adipose and muscular tissues, including the heart, whose activity depends heavily (up to 80 % in certain physiological conditions) on the oxidation of fatty acids.

 

Chylomicron metabolic process allows the understanding of the causes and characteristics of Hiperlipoproteinemia Tipe I, familial hyperchylomicronemia or familial lipoprotein lipase deficiency, whose main feature is an increase of  chylomicron concentration and a very slow plasma clearence of them. The genetically identified causes of this disease are the deficit of Liporptein Lipase, the production of an abnormal LPL, or the deficit of Apo C-II  (in that case, LPL would not be activated. Clinical features are abdominal pain afer the ingestion of fat-rich meals,  xanthomas, acute pancreatitis, hepatic steatosis, as result of an excess of fatty deposit in various tissues.

 

Related links

 

http://en.wikipedia.org/wiki/Chylomicrons

 

http://themedicalbiochemistrypage.org/lipoproteins.html#uptake

 

http://themedicalbiochemistrypage.org/lipoproteins.html#chylomicrons

 

Related Posts:

 

Structure and Classification of Lipoproteins

 

Metabolism of VLDL and Formation of IDL

 

Apolipoproteins

 

Q: About Enzyme Regulation


Enzyme Question (E-09)

 

A Protein Kinase

A Protein Kinase

 

 

 

 

 

 

Protein kinases are enzymes that act on other enzymes by adding phosphates groups. When the enzyme is phosphorylated, it changes its activity (it becomes more or less active, depending on the enzyme). This regulatory mechanism of enzymatic activity is called:

 

a)     Allosteric Control

 

b)     Competitive inhibition

 

c)      Covalent Modification

 

d)     Isozymes Modification

 

e)     Zymogen activation

 

Some questions about Lipoproteins


(LM-06) This kind of lipoprotein is synthesized in the intestinal cells and releases fatty acids in muscle and adipose tissue.

 

a)     Chylomicrons

b)     HDL

c)      IDL

d)     LDL

e)     VLDL

 

(LM-07)Lipoprotein lipase acts in:

 

a)     Hydrolysis of triacylglycerols of plasma lipoproteins to supply fatty acids to various tissues.

b)     Intestinal uptake of dietary fat

c)      Intracellular hydrolysis of lipids from adipose tissue

d)     Hydrolysis of amino acids from lipoproteins

e)     Digestion of lipids contained in foods

 

(LM-08) This apoprotein is necessary for the activation of lipoprotein lipase:

 

a)     Apo A-I

b)     Apo A-II

c)      Apo A-IV

d)     Apo B-48

e)     Apo B-100

f)       Apo C-I

g)     Apo C-II

h)    Apo C-III

i)       Apo D

j)       Apo E

 

Answer

 

(LM-09) Fatty liver (hepatic steatosis) occurs in some conditions as obesity, uncontrolled diabetes mellitus and chronic ethanol ingestion, in which there is an imbalance between hepatic triacylglycerol synthesis and the secretion of the lipoproteins that transfer hepatic triacylglycerols and other lipids to peripheral tissues. These lipoproteins are:

 

f)     Chylomicrons

g)     HDL

h)     IDL

i)     LDL

j)     VLDL

 

 

Apolipoproteins: the apoproteins in lipoproteins


 

In a wide sense, apoprotein is the term applied to the proteinic part (the one formed by amino acids) of conjugate proteins.

 

In a more narrow sense, and the one usually used in medical practice, apoprotein is the proteic part of a lipoprotein. From the technical point of view, it is more appropriate to call them “apolipoproteins”  

 

A common “motif” in the structure of apolipoproteins is the presence of amphipatic helixes that allow the interaction with the aqueous environment that surrounds the lipoprotein, and with the lipid interface of the lipoprotein.

 

In general, the apolipoproteins, besides contributing to the solubility of the lipoproteins as a whole, act as ligands for cellular receptors and as cofactors of enzymes related to the lipoprotein metabolism.

 

Apolipoproteins have been classified in families according to the size, distribution in lipoproteins and other characteristics.

 

The following table shows some important apolipoproteins, whose functions are known:

 

Apolipoprotein

Molecular Weight

Important Features

Apo A-1

29 000

Main protein in HDL; activates LCAT

Apo B-100

513 000

Main protein in LDL; it binds to LDL receptor

Apo C-II

8 800

Important in the composition of Chylomicrons and VLDL; activates Lipoprotein Lipase.

Apo E

34 000

Important in Chylomicrons, VLDL and IDL, allowing the binding of these lipoproteins to the hepatocytes.

 

 

More information about Apoproteins:

 

http://themedicalbiochemistrypage.org/lipoproteins.html#classification

 

 Related Posts:

 

Structure and Classification of Lipoproteins

 

 Metabolism of VLDL and Formation of IDL

 

 

Metabolism of Chylomicrons

 

 

 

Q: About Collagen Synthesis


Question about Proteins P-07

 

Hydroxyproline

Hydroxyproline

 

Collagen presents in its structure modified amino acids as hydroxyproline and hydroxylysine. The formation of these amino acids from their precursors, is post-trancriptional, and occurs in enzymatic reactions that require as cofactor the following compound:

 

a)     Ascorbic acid

 

b)     Citric Acid

 

c)      Folic Acid

 

d)     Lipoic acid

 

e)     Panthotenic acid