Sugar nucleotide role in metabolism Glycogen synthesis and breakdown Ferchmin 2017 Sugar nucleotide role in metabolism Glycogen synthesis and breakdown 2) Introduction 3) Synthesis of sugar nucleotides 4-5) Examples of the function of activated monosaccharides 6-7) Galactose metabolism. Galactosemia types 1, 2, and 3. 8) Lactose 9-11) Hyperglycemia causes protein modifications 12-16) Glycogen synthesis and breakdown 17-18) Glycogen storage diseases (GSD)

Introduction The intermediates in glycolysis, pentose shunt, and gluconeogenesis are sugar phosphate. However, in many metabolic steps sugars are activated as nucleotides where the sugar is bond to a nucleotide through the anomeric hydroxyl as a phosphate ester. Sugar nucleotides are the substrates for polymerization into disaccharides, glycogen, and complex homo- or hetero-polysaccharides. They are also key intermediates in the metabolism of some sugars. Sugar nucleotides not only have the energy of the pyrophosphate bond that is needed to bind covalently the monosaccharide to a different molecule but also the nucleotide is a label that defines a different sugar pool with a specific function. Enzymes will recognize as substrate either the free monosaccharide or the nucleotide. In other words, free glucose, phosphorylated glucose and UDP-glucose are different molecules destined to perform a particular function. The role of sugar nucleotides (specifically UDP-glucose) in the biosynthesis of glycogen and many other carbohydrate derivatives was discovered by Noble Laureate Dr. Luis Federico Leloir.

Several nucleotides can activate sugars Several nucleotides can activate sugars. In the case of glucose for glycogen synthesis it is UTP. The first reaction a), is reversible but it is coupled to b) that releases -5 kcal/mol and favors reaction a).

Sugar nucleotides of glucose or of any other carbohydrate are called activated because sugars bound to nucleotides can be transferred by specific enzymes to proteins and other molecules or can be subjected to enzymatic modifications possible only with activated sugars. UDP-glucuronic is important for detoxification of many drugs and metabolites. Below is shown the formula of conjugated bilirubin: We, primates, lost the ability to make ascorbate. Therefore we are genetically deficient.

Galactose metabolism Leloir pathway Note: these curved arrow lines were supposed to be bidirectional to show the reversibility of the reactions. Also, the lines crossover reflecting the exchange of “partner” between glucose and galactose. Fructose is an ketose, therefore it is not a substrate of aldose reductase and causes no cataracts. Glucose and galactose are aldoses and they do cause cataracts Deficiency of galactokinase causes a mild form of galactosemia that causes cataracts. Deficiency of hexose-1-phosphate-uridylyl transferase causes the sever type of galactosemia with liver failure, mental retardation and cataracts. The name galactose-1-phosphate-uridylyl transferase (in drawing) is simply wrong but commonly used including in the NBE. The correct name is hexose-1-phosphate uridylyl transferase. This is so because it transfers equally well glucose as it does galactose. Therefore, the name in the fig is not in agreement with the commission for enzyme nomenclature but it is used in most books and in the NBE. The 4 epimerase uses NAD+ as cofactor and the transition state is 4 keto hexose. Type III galactosemia is caused by mutations in the gene encoding UDP-glucose-4-epimerase. A variety of different point mutations located throughout the gene is responsible. The main, disease-causing effects of these mutations appear to be a reduction in the catalytic rate constant and instability of the protein. Galactosemia III is very rare and the symptoms vary depending on the mutation(s).

Galactose metabolism Leloir pathway Fructose is an ketose, therefore it is not a substrate of aldose reductase and causes no cataracts. Glucose and galactose are aldoses and they do cause cataracts This slide is a repetition without at annoying clarification about the reversibility and the arrows. Deficiency of galactokinase causes a mild form of galactosemia that causes cataracts. Deficiency of hexose-1-phosphate-uridylyl transferase causes the sever type of galactosemia with liver failure, mental retardation and cataracts. The name galactose-1-phosphate-uridylyl transferase (in drawing) is simply wrong but commonly used including in the NBE. The correct name is hexose-1-phosphate uridylyl transferase. This is so because it transfers equally well glucose as it does galactose. Therefore, the name in the fig is not in agreement with the commission for enzyme nomenclature but it is used in most books and in the NBE. The 4 epimerase uses NAD+ as cofactor and the transition state is 4 keto hexose. Type III galactosemia is caused by mutations in the gene encoding UDP-glucose-4-epimerase. A variety of different point mutations located throughout the gene is responsible. The main, disease-causing effects of these mutations appear to be a reduction in the catalytic rate constant and instability of the protein. Galactosemia III is very rare and the symptoms vary depending on the mutation(s).

Lactose synthesis is mediated by a galactosyl transferase that binds galactose with N-acetyl-glucosamine to give N-acetyllactosamine, a constituent of several oligosaccharides. At the onset of lactation a protein specific to the mammary gland increases the affinity of this enzyme for glucose from 1 molar to 1 mM. Lactose is metabolized by LACTASE or β-galactosidase. This enzyme should be lost after weaning. However, many ethnic groups keep β-galactosidase for life. The biology of lactose is very interesting and clinically relevant. A clinical exercise about lactose intolerance used to be done before .You will have to do it as a self study. ß1,4 Galactosyltransferase is unique among all glycosyltransferases in that its substrate specificity can be modified by addition of a-lactalbumin. Together, ß1,4 galactosyltransferase and a-lactalbumin form the lactose synthase complex. Because a-lactalbumin is only expressed in the mammary gland, lactose synthesis only occurs in the mammary gland. In addition, expression of the a-lactalbumin gene is closely regulated by hormones, so that lactose synthesis only occurs during the lactating state of the tissue.

Excess of glucose can deplete NADPH+H+ and cause opacity of the lens (cataracts). Conversion of glucose to sorbitol with ensuing osmotic disturbance.  This occurs through the "polyol pathway", a normal reaction sequence in testes but not other tissues.  In the first step, Glucose is converted to sorbitol by aldose reductase.  The sorbitol formed is then oxidized to fructose. A problem arises when sorbitol production occurs in tissues other than testes. At high glucose levels, aldose reductase activity occurs in other organs which often lack sorbitol dehydrogenase. This leads to an accumulation of sorbitol in these tissues, notably in the lens of the eye, leading to cataract formation due to osmotic damage. The sperm gets its fructose through this pathway. Why the sperm needs fructose and not glucose? Sorbitol (and galactitol) cause cataracts and osmotic disturbances

Glycosylation (glycation) of hemoglobin and formation of HbA1c Glycation (sometimes called non-enzymatic glycosylation) is the result of the covalent bonding of a sugar molecule, such as glucose or fructose, (with a potentially free carbonyl grop) to a protein or lipid molecule, without the controlling action of an enzyme. The degree of glycation of the body's proteins is related to blood glucose levels.  Hemoglobin has a half-live of about 100 days.  The degree of glycation of hemoglobin gives, therefore, a picture of average blood glucose levels for the previous three month period.   Glycated hemoglobin is known as HbA1c.  Normally, HbA1c accounts for approximately 5-6 % of the total hemoglobin.  Diabetic patients often have HbA1c in excess of 8-10%.  It has been usually assumed that glycation of hemoglobin followed reaction with glucose, as shown in the next figure.  Current studies have, as noted above, indicated that other reactive species, especially methylglyoxal, may be involved in this process.  Note that the degree of glycation of hemoglobin is an indication of the extent of glycation of many of the body's proteins and possible ensuing cellular damage.  How is it possible for a diabetic patient to have abnormally low HbA1C? Will you be good at making diagnosis?

Recapitulation of cataract, sugar alcohols protein glycosylation Glucose, generates by no-enzymatically glyoxal and methylglyoxal with two carbonyl groups which react with proteins, RNA and DNA to form stable addition products. This is non-enzymatic glucosylation (or glycation) is a normal process which follows reaction of the carbonyl compounds with.  The process and the rate of reaction is proportional to the concentration of glucose in the blood.   The glycosylated proteins or nucleic acids undergo cross-linking, forming "AGE" or advanced glycation end-products.  There are several enzymes that protect the cell by removing the glycosylation groups and there seem to be a race between the glycosylation and the removal of the bound harmful groups. This is a hot topic for diabetes, aging, and other medical problems but I do not know whether it is reflected in the NBE

Glycogen Metabolism

Synthesis of uridinediphosphoglucose or UDPGlu Synthesis of Glycogen Synthesis of uridinediphosphoglucose or UDPGlu Do you remember phosphoglyceromutase? Any similarities with phosphoglucomutase? PPi is hydrolyzed by a pyrophosphorylase in a reaction coupled with the pyrophosphorylase to dissipate energy as heat thus making the synthesis of UDP-Glu thermodynamically favorable.

Why do we need to waste 2 ATPs and make glycogen? Deficiency of branching enzyme gives long branches. Causes death at about to years of age. Andersen’s disease The content of glycogen is about 10 % of the wet weight of the liver and 2% of muscle.

Glycogen synthase only adds glucoses to a pre-existing chain of at least 4 glucose residues. Glycogenin acts by catalyzing the addition of glucose to itself (autocatalysis) by first binding glucose from UDP-glucose to the hydroxyl of Tyr-194 from UDP-glucose, by glycogenin's glucosyltransferase. Once sufficient residues have been added, glycogen synthase takes over extending the chain. Glycogenin remains covalently attached to the reducing end of glycogen.

Glycogen degradation The breakdown of glycogen and entry into glycolysis as glucose-6-P is achieved by three enzymes: glycogen phosphorylase, debranching enzyme and phosphoglucomutase. Glycogen phosphorylase produces glucose-1-P plus limit dextrin. The debranching enzyme has a transferase and glycosidase (hydrolase) activities. Hexokinase is bypassed when glucose comes from glycogen! Deficiency of phosphorylase (Mc Adler’s) causes muscle cramps and no lactate formation during exercise. Deficiency of debranching enzyme causes accumulation of limit dextrin

There are 10 (ten) glycogen storage diseases (GSD) although there is no consensus about the exact number of GSD. I will not lecture specifically about the GDS but you are responsible to study them. The most clinically oriented and stress-free site to study GSDs is the webpage of the Association for Glycogen Storage Disease. The link takes you directly to relevant information. http://www.agsdus.org/what-is-gsd.php In the next “handout” we will begin with regulatory mechanisms involved in glycogen metabolism