Question

1. Try to answer the following questions as accurately as possible

a) There are about 15 or so mechanisms by which metabolism can be controlled at different levels. which are the 15 mechanisms by which metabolism can be controlled?

b) identify three mechanisms that can control metabolism with immediate effect

c) identify three mechanisms that require more time to exert their effects

d) Humans have 4 isoenzymes that differ in how they function. why do we need 4 isozymes?

e)  How are phosphofructokinase-1 and fructose 1,6-bisphosphatase regulated?

f) 7 reactions in glycolysis are used in the reverse direction in gluconeogenesis. How can this be?

Answer 1

(a)Basic metabolic regulation mechanisms are catabolite regulation, nitrogen regulation, and phosphate regulation, as well as the effects of acidic pH, heat shock, and nutrient starvation on metabolic regulations. Attention focuses on the effects of global regulators (transcription factors with sigma factors), such as cAMP-Crp, Cra, Mlc, RpoN, ArcA/B, Fnr, SoxR/S, PhoR/B, RpoH, and RpoS on metabolism. The effects of knockout of such genes as cra, crp, mlc, arcA/B, phoR/B, soxR/S, and rpoS.

Main Metabolism:-

Metabolic Regulation and futile Cycle :-

Metabolic regulation of a cell achieves cell growth by optimizing ATP generation (catabolism) and cell synthesis (anabolism) during the cell growth phase. Moreover, the cell regulates the metabolism to cope with various kinds of stresses caused by changes in the culture environment, and thus it is not easy to understand the whole metabolic regulation mechanism. Metabolic regulation occurs at both gene and enzyme levels, where enzyme level regulation is typically made by allosteric regulation, which is attained by changing the 3D structure by binding the specific metabolites, etc. For example, G6PDH and 6PGDH are inhibited by NADPH, while Pfk is inhibited by PEP. PDH is also inhibited by NADH, ATP, AcCoA, and so on.

Metabolic regulation is the physiological mechanism by which the body takes in nutrients and delivers energy as required. Metabolic regulation works ultimately at a molecular level, mainly by modulation of enzyme activities that function together as a whole system to sense the balance of energy coming in and energy required. The different organs in the body have their own characteristic patterns of metabolism according to their functions in the body. Thus, it is critical that metabolic pathways interact in a dynamic sense, in the entire organism. Furthermore, the endocrine and nervous systems need to precisely coordinate to control the flow of energy within the body.

Much of the metabolic regulation is governed by hormones that are delivered through the bloodstream and act through specific cellular receptors. Both the cell-surface receptors (that usually bind peptide hormones) and the nuclear receptors (that bind thyroid hormones, steroid hormones, and other membrane-permeant ligands) play critical roles in metabolic regulation. Hormones acting through cell-surface receptors are involved in rapid metabolic adjustments. These receptors signal via the small molecule cyclic adenosine 3,5-monophosphate (cyclic AMP or cAMP) and the membrane lipid phosphatidylinositol (3,4,5) trisphosphate. Following this, the activities of downstream metabolic enzymes are regulated by covalent modification, especially phosphorylation and dephosphorylation, and/or translocation of enzymes within the cell. Activation of the nuclear receptors by their ligands, on the other hand, directly controls the transcription of metabolic genes and leads to long-term metabolic regulation.

(b)

1. To produce products only when needed.

2. There are lots of pathways in the cell, some of which are opposing in direction (one produces a compound, the other pathway metabolizes that compound). In general, opposing pathways do not occur at the same rate, at the same time in the same cell. If they did, then no net product would be produced, but in many cases ATP and/or NADH would be consumed. When this happens, it is called complete futile cycling. Regulating enzyme activity prevents complete futile cycling and prevents the synthesis of unneeded or excess products. However, partial futile cycling is very common and useful in regulating flux.

Exceptions: heat generation, ATP hydrolysis releases energy as heat

Partial exception: having opposing steps occur at the same time allows greater control over the flux

IN GENERAL: To prevent complete futile cycling, enzymes of opposing irreversible reactions are regulated in opposite directions. This also allows greater change in flux.

How is metabolic flux regulated?

A. Regulate amount of Substrate

B. Regulate activity of Enzyme via changes in:

1. Enzyme amount (synthesis vrs degradation)

2. Type of enzyme present- (isozymes with different catalytic properties)

3. Enzyme specific activity (see below), changing activity of the same amount of enzyme.

A major mechanism of enzyme regulation is regulating enzyme specific activity

There are two main types of mechanisms for regulation of enzyme specific activity:

1 .Allostery-Enzyme activity can be activated or inhibited through non-covalent interaction of the enzyme with small molecules called effectors, modulators, or allosteric regulators (AE). Effectors bind to the enzyme at a site other than the active site (allo means other). Reversible binding to enzyme- fast response time. Concentration dependent.

2. Covalent modification- Covalent addition of a group to an enzyme changes the activity of the enzyme.

Mechanisms of Reciprocal Regulation:

To prevent complete futile cycling, regulation of enzyme activity by allostery or covalent modification works reciprocally. That is, the specific activities of one or more enzymes of a pathway are activated at the same time the specific activity of one or more enzymes of the opposing pathway is inhibited. Often the same mechanism is used (either the same modification system, or the same allosteric regulator). The same mechanism has opposite effects on enzymes of the opposing pathways.

Reciprocal regulation using allostery- Binding of the same regulator to the enzymes of opposing reactions has the opposite effect (activation for one enzyme, inhibition for the other).

Reciprocal regulation using covalent modification- The modification system modifies enzymes catalyzing opposing reactions at the same time, the same type of modification has opposite effects on the two enzymes, activating one, while inhibiting the other that operates in the opposing pathway.

(d)

Isoenzymes

Isoenzymes (also called isozymes) are alternative forms of the same enzyme activity that exist in different proportions in different tissues. Isoenzymes differ in amino acid composition and sequence and multimeric quaternary structure; mostly, but not always, they have similar (conserved) structures. Their expression in a given tissue is a function of the regulation of the gene for the respective subunits. Each isoenzyme form will have different kinetic and/or regulatory properties that reflect its role in that tissue. Isoenzymes are generally identified in the clinical laboratory by electrophoresis.

Isozymes

Isozymes (or isoenzymes) catalyze similar reactions, but differ from each other slightly in chemical structure, and therefore kinetic properties. They are usually organ-specific, and therefore exploited for diagnostic purposes. Most enzymes present in plasma are released during normal cell turnover. However, increased amounts of particular isozymes appearing in plasma usually reveal trauma or pathologic processes. They are usually separated from each other in the diagnostic laboratory via electrophoresis.

Creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) are two isozymes commonly used for diagnostic purposes (Table 6-2). The CPK enzyme (see Chapter 77) contains two dimers assembled from muscle (M) and brain (B) subtypes. Skeletal muscle CPK is almost entirely the MM isozyme, brain BB, and cardiac muscle 15% MB and 85% MM (Fig. 6-5). Only cardiac muscle has the MB dimer. Lactate dehydrogenase is a tetramer, assembled from M and heart (H) subunits, with 5 different isozymes appearing via electrophoresis. Following a myocardial ischemic episode, LDH release into plasma usually occurs after that of CPK and troponin-I. The troponins (Tn-I, Tn-T and Tn-C) constitutue a regulatory complex of globular muscle proteins of the I band that normally inhibit contraction by blocking interaction of actin and myosin. Another polypeptide from ventricular myocytes, N-terminal-pro-Brain Natriuretic Peptide (NT-proBNP), is usually elevated in cardiac hypertrophy (or dilation) in animals with congestive heart failure.

Table 6-2. Examples of Serum Enzymes Used for Diagnostic Purposes

Enzyme	Purpose
Alanine aminotransferase	Hepatic necrosis in dogs & cats.
Alkaline phosphatase	Bile duct obstruction and bone disease.
Amylase	Acute pancreatitis.
Aspartate aminotransferase	Liver disease in large animals; myopathies, hemolysis, and intestinal disease.
Creatine phosphokinase	Myopathies (& myocardial infarction).
Lactate dehydrogenase	Myocardial infarction; brain, kidney, pancreatic, lung, spleen, liver and erythrocyte disorders.
Lipase	Acute pancreatitis.
Sorbitol dehydrogenase	Liver disease in large animals.

(e)

Phosphofructokinase

The key regulatory enzyme of glycolysis is phosphofructokinase. It is inhibited by ATP and citrate and activated by AMP (and ADP), Pi, and fructose 2,6-bisphosphate. Although ATP is a substrate of the enzyme, it is also an important product of glycolysis, and the inhibition at a regulatory site is an example of classic feedback inhibition. Since ATP usage produces AMP, ADP, and Pi, these activators also signal a need for more ATP production, as would a decrease in ATP. Citrate may serve as an indicator of the sufficiency of alternative fuel, particularly fatty acids that are broken down to acetyl-CoA, which may then increase citrate so that glucose may be preserved for the brain. The importance of fructose 2,6-bisphosphate as an activator has principally been established in liver; it is discussed later in the context of reciprocal regulation of gluconeogenesis and glycolysis. (It is made by phosphorylation of fructose 6-phosphate on the 2 position by a phosphofructokinase-2, distinct from the glycolytic phosphofructokinase.)

Phosphorylation of Fructose-6-Phosphate

In the third step, phosphofructokinase-1 (PFK-1) transfers the phosphate residue of ATP to the C1 hydroxyl residue of F6P, generating fructose-1,6-bisphosphate (F1,6BP). This reaction requires Mg2+ and fructose-2,6-bisphosphate. After ingestion of a meal, fructose-2,6-bisphosphate is elevated, resulting in the above-mentioned reaction advancing a speedy glycolysis reaction. Before a meal, the concentration of fructose-2,6- bisphosphate is decreased, resulting in upregulation of F1,6BP and facilitation of gluconeogenesis. Although G6P and F6P are also metabolized in pathways other than the glycolytic system, F1,6BP is metabolized only by the glycolytic pathway. The reaction in which PFK-1 acts as a catalyst is irreversible, and PFK-1 does not use the reverse reaction in the case of glyconeogenesis. Therefore, two enzyme reactions modulated by fructose- 2,6-bisphosphate are a key point of the glycolytic and/or glyconeogenesis system.

(f) Glucogenesise :- it is the metabolic process which is used by organisms produce suar namely glucose for catabloix reaction from non- carbohydates precusrsor.  Glucose is the only energy source used by the brain (with the exception of ketone bodies during times of fasting), testes, erythrocytes, and kidney medulla. In mammals this process occurs in the liver and kidneys.

Overview

Gluconeogenesis is much like glycolysis only the process occurs in reverse. However, there are exceptions. In glycolysis there are three highly exergonic steps (steps 1,3,10). These are also regulatory steps which include the enzymes hexokinase, phosphofructokinase, and pyruvate kinase. Biological reactions can occur in both the forward and reverse direction. If the reaction occurs in the reverse direction the energy normally released in that reaction is now required. If gluconeogenesis were to simply occur in reverse the reaction would require too much energy to be profitable to that particular organism. In order to overcome this problem, nature has evolved three other enzymes to replace the glycolysis enzymes hexokinase, phosphofructokinase, and pyruvate kinase when going through the process of gluconeogenesis:

1. The first step in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvic acid (PEP). In order to convert pyruvate to PEP there are several steps and several enzymes required. Pyruvate carboxylase, PEP carboxykinase and malate dehydrogenase are the three enzymes responsible for this conversion. Pyruvate carboxylase is found on the mitochondria and converts pyruvate into oxaloacetate. Because oxaloacetate cannot pass through the mitochondria membranes it must be first converted into malate by malate dehydrogenase. Malate can then cross the mitochondria membrane into the cytoplasm where it is then converted back into oxaloacetate with another malate dehydrogenase. Lastly, oxaloacetate is converted into PEP via PEP carboxykinase. The next several steps are exactly the same as glycolysis only the process is in reverse.
2. The second step that differs from glycolysis is the conversion of fructose-1,6-bP to fructose-6-P with the use of the enzyme fructose-1,6-phosphatase. The conversion of fructose-6-P to glucose-6-P uses the same enzyme as glycolysis, phosphoglucoisomerase.
3. The last step that differs from glycolysis is the conversion of glucose-6-P to glucose with the enzyme glucose-6-phosphatase. This enzyme is located in the endoplasmic reticulum.

4. Regulation

   Because it is important for organisms to conserve energy, they have derived ways to regulate those metabolic pathways that require and release the most energy. In glycolysis and gluconeogenesis seven of the ten steps occur at or near equilibrium. In gluconeogenesis the conversion of pyruvate to PEP, the conversion of fructose-1,6-bP, and the conversion of glucose-6-P to glucose all occur very spontaneously which is why these processes are highly regulated. It is important for the organism to conserve as much energy as possible. When there is an excess of energy available, gluconeogenesis is inhibited. When energy is required, gluconeogenesis is activated.

5. The conversion of pyruvate to PEP is regulated by acetyl-CoA. More specifically pyruvate carboxylase is activated by acetyl-CoA. Because acetyl-CoA is an important metabolite in the TCA cycle which produces a lot of energy, when concentrations of acetyl-CoA are high organisms use pyruvate carboxylase to channel pyruvate away from the TCA cycle. If the organism does not need more energy, then it is best to divert those metabolites towards storage or other necessary processes.
6. The conversion of fructose-1,6-bP to fructose-6-P with the use of fructose-1,6-phosphatase is negatively regulated and inhibited by the molecules AMP and fructose-2,6-bP. These are reciprocal regulators to glycolysis' phosphofructokinase. Phosphofructosekinase is positively regulated by AMP and fructose-2,6-bP. Once again, when the energy levels produced are higher than needed, i.e. a large ATP to AMP ratio, the organism increases gluconeogenesis and decreases glycolysis. The opposite also applies when energy levels are lower than needed, i.e. a low ATP to AMP ratio, the organism increases glycolysis and decreases gluconeogenesis.
7. The conversion of glucose-6-P to glucose with use of glucose-6-phosphatase is controlled by substrate level regulation. The metabolite responsible for this type of regulation is glucose-6-P. As levels of glucose-6-P increase, glucose-6-phosphatase increases activity and more glucose is produced. Thus glycolysis is unable to proceed.